The Role of Superoxide Dismutase in Regulating the Light Emission of Luminescent Fungi

Luminescent fungi spontaneously emit light during certain stagesof their life cycles. Most of them are luminous during a partof their mycelial stage, but not many of them are luminous whenthey form fruiting bodies. In the case of Panellus stipticus,both the mycelium and the fruiting body can be luminous, andthe emission of light takes place when its luciferin is aerobicallyoxidized in the presence of the superoxide anion (O₂) and acationic surfactant. It is highly likely that the luminescencereactions of all kinds of luminous fungi are basically the sameas that of P. stipticus. In order to determine the factor thatmakes a fungus luminous or non-luminous, we studied the relationsbetween the light emission of fungi at various growth stagesand the contents of luciferin, its precursor, superoxide dismutase(SOD), and catalase, on six species of luminescent fungi: Armillariellamellea, Mycena citricolor, Mycena lux-coeli, Omphlotus olearious,Panellus stipticus, and Pleurotus japonicus. The analysis ofthe data suggested that the fungi generally contain the componentsnecessary for light emission, but also contain very large amountsof SOD which destroy O₂^–. If an appreciable amount ofSOD is distributed at the site of light emission, the luminescencereaction is prevented. For the reaction to take place, it isessential that the SOD activity at the site is sufficientlylow or inhibited, despite the high content of SOD in the wholetissue. Thus, the level of SOD activity at the site of lightemission appears to be a limiting factor in regulating the luminescenceof fungi. Key words: Bioluminescence, chemiluminescence, luminous fungi, superoxide ion, superoxide dismutase     

Structure and non-enzymatic light emission of two luciferin precursors isolated from the luminous mushroom Panellus stipticus

The chemical structure of two luciferin precursors PS-A and PS-B, isolated from the luminous mushroom Panellus stipticus, were determined as 1-O-decanoylpanal (2) and 1-O-dodecanoylpanal (3), respectively. Both PS-A and PS-B were converted into chemiluminescent luciferins by treatment with 50 mmol/l methylamine in a pH 3.5 buffer solution containing an anionic surfactant Tergitol 4 at 25–35ºC. The luciferins emitted chemiluminescence in a pH 7–8 buffer solution containing a cationic surfactant in the presence of O₂ and O.     

Determination of phenol using an enhanced chemiluminescent assay

Enhanced chemiluminescence (ECL) describes the phenomenon of increased light output in the luminol oxidation reaction catalysed by horseradish peroxidase (HRP) in the presence of certain compounds, such as para‐iodophenol. In this work, the effects of phenol on the para‐iodophenol‐enhanced HRP‐catalysed chemiluninescent reaction intensity in an aqueous buffer (Tris–HCl buffer, pH 8.5) and in a surfactant–water–octane mixture were compared. Preincubation of HRP at low phenol concentrations stimulated the chemiluminescent intensity in the assay performed in an aqueous buffer, but did not have significant effect in the sodium bis(2‐ethylhexyl)sulphosuccinate) (Aerosol OT, AOT) applied system. It was also observed that HRP preincubation with phenol concentration higher than 0.003 mg/mL produced an inhibitory effect on the enzyme activity for both assay systems. Only an inhibitory effect of phenol on the chemiluminescent intensity in the surfactant system in octane (as organic solvent) was observed. Three assays were developed to determine phenol concentration in water and in an organic solvent mixture. The detection limits were 0.006, 0.003 and 0.0005 mg/mL, respectively, for the buffer‐containing system, the AOT‐applied system with phenol standard solutions in water and for the AOT‐applied system with phenol standard solutions in octane. Copyright © 2002 John Wiley & Sons, Ltd.     

Occurrence of mycosporine-like amino acids in the red-tide dinoflagellate Alexandrium excavatum: UV-photoprotective compounds?

Water extracts of the red-tide dinoflagellate Alexandrium excavatumgrown at ‘high’ light intensity (200 µE m^–2s^–1) show a broad absorbance maximum in the UV regionof the spectrum (310–360 nm). Using TLC and reverse-phaseHPLC a series of mycosporine-like amino acids have been characterized:mycosporine-glycine (_max = 310 nm), palythine (_max = 320 nm),asterina-330 (_max = 330 nm), shinorine (_max = 334 nm), porphyra-334(_max= 334 nm), palythenic acid (_max = 337 nm) and the isomericmixture of usujirene and palythene (_max = 359 nm). From theobserved spectral changes during transference from ‘low’(20 µE m^–2 s^–1) to ‘high’ (200µE m^–2 s^–1) light intensities and vice versa,the series of compounds are supposed to be biogenically relatedto one another. The presence of these compounds in A.excavatumis discussed in relation to their possible role in the photoprotectionto deleterious UV radiation.     

Red‐shifted emission from 1,2‐dioxetane‐based chemiluminescent reactions

Commercial chemiluminescent reagents emit across a broad portion of the electromagnetic spectrum (400–500 nm). A challenge to the use of chemiluminescence to monitor biological processes is the presence of interfering substances in the biological optical window. In the present study, longer wavelength emitting fluorophores (the organic dyes Alexa 568 and Alexa 647), and a semiconductor nanoparticle (QDOT800) were used to red‐shift the emission from commercially available 1,2‐dioxetane‐based chemiluminescent substrate reactions. By adding non‐conjugated fluorescent emitters into chemiluminescent reaction mixtures, an emission peak occurred at the predicted wavelength of the fluorescent emitter. The excitation and emission from QDOT800 was preserved in the presence of a 100 µm‐thick glass barrier separating it from the chemiluminescent reaction components. The maximum tissue phantom penetration by QDOT800 emission was 8.5 mm; in comparison, the native chemiluminescent emission at 500 nm was unable to penetrate the thinnest tissue phantom of 2.5 mm. The described method for red‐shifted emissions from chemiluminescent reactions does not require direct interaction between the chemiluminescent reaction and the fluorescent emitters. This suggests that the mechanism of chemiluminescent excitation of fluorophores and QDOT800 is not exclusive to chemiluminescence resonance energy transfer or sensitized chemiluminescence, but rather by broad energization from the native chemiluminescent emission. Copyright © 2014 John Wiley & Sons, Ltd.     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

Phytochrome in the Fern, Adiantum capillus-veneris L.: Spectrophotometric Detection in Vivo and Partial Purification

Spectrophotometric studies of fern phytochrome were performedusing dark-grown leaves of Adiantum. The absorbance differencespectrum between the red- and far-red-light irradiated sampleshowed a photoreversible absorbance change in the far-red region,with a maximum located at 728–730 nm. The concentrationof phytochrome was highest at the leaf tips and decreased graduallyalong the leaf axis. As in the case of angiosperm phytochrome,the level of fern phytochrome decreased under continuous whitelight, and the level increased again when deetiolated tissuewas transferred back to the dark. When the fern tissue was exposedto a pulse of red light, the dark reversion of P_FR to P_R tookplace with almost no destruction of P_FR. Phytochrome could beextracted from light-grown young leaves of the fern with a slightlyalkaline, aqueous buffer that contained 1 M NaCl. The differencespectrum of the partially purified phytochrome from fern wassimilar to that of partially degraded phytochrome from angio-sperms.A polyclonal antibody raised against phytochrome from etiolatedrye seedlings immuno-stained (albeit weakly) a 110-kDa polypeptideafter fractionation by SDS-polyacrylamide gel electrophoresisof the preparation of fern phytochrome. The band was very probablyfern phytochrome since it emitted zinc-induced fluorescence. (Received July 12, 1990; Accepted October 5, 1990)     

In vivo-Microspectrophotometric Characterization of Flavonol Glycosides in Vicia faba Guard and Epidermal Cells

Cytological observations by fluorescence and U.V.-absorptionmicroscopy together with in vivo spectrophotometric analysesof stomata, guard cell protoplasts and epidermal cells of Viciafaba have shown that kaempferol 3,7-O-glycosides are localizedin the vacuoles. The alkaline-induced emission spectra recordedwith guard and epidermal cells after NH₄OH-treatment were identical,exhibiting an emission maximum at 525 nm; the spectra correlatedwith that of reference flavonols after exposure to NH₄OH Theexcitation spectra of both cell types are typical of these flavonolsshowing two maxima at 290 nm and 390 nm. In agreement, two absorptionmaxima were recorded for guard cells at 270 nm and 330 nm, withoutalkali, which shifted bathochromically to 275 nm and 380 nm,respectively, after NH₄OH treatment. The fluorescence intensitymeasured at 525 nm demonstrates a photostability in epidermalcells whereas it increases by a factor of about five with theexcitation time up to 30 min in guard cells. For the latter,several possible processes are discussed. Key words: Alkaline-induced fluorescence, emission, excitation, U.V.-absorption spectra, kaempferol glycosides, cell specificity     

The effect of UV-B radiation and humic substances on growth and motility of the flagellate, Euglena gracilis

The effects of ultraviolet-B radiation (UV-B, 250–315nm) were determined on Euglena gracilis with respect to speed,phototactic orientation, specific growth rate, and in the presenceof humic substances. Humic substances had a protective effectwhen studying the speed and specific growth rate. However, thedegree of phototactic orientation decreased in UV-B radiationboth with and without humic substances. The inhibition of O₂evolution and speed was most pronounced when using cutoff filtersWG280 and WG295. The photosynthetic inhibitor DCMU (10^–6M) did not have any effect on the speed, but the 0₂ evolutiondecreased to zero. The effect of different wavelengths in theUV-B region on the speed of E.gracilis showed the maximum sensitivityat 280 and 290 nm.     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

Time-Resolved Spectroscopy of Chlorophyll-a like Electron Acceptor in the Reaction Center Complex of the Green Sulfur Bacterium Chlorobium tepidum

The absorption changes of chlorophyll (Chl) a-like pigments(C670) were studied by ns-ms laser spectroscopy at 77 K in theuntreated and urea-treated homodimeric reaction center (RC)complex of the green sulfur bacterium Chlorobium tepidum. Theuntreated RC complex contained 9 molecules of C670 in additionto 41 molecules of Bchl a and 0.9 molecules of menaquinone-7per one primary electron donor Bchl a dimer (P840). Upon photo-oxidationof P840, C670 showed an absorption change of a red-shift withan isosbestic wavelength at 668 nm. The absorption change ofP840 decayed with time constants (t_1/e) of 55 and 37 ms at 283and 77 K, respectively, and was assigned to represent the chargerecombination between P840⁺ and FeS^–. In the urea-treatedRC complex, a bleach peaking at 670 nm with a shoulder peakat 662 nm, which is ascribable to the reduced primary electronacceptor A₀^–, was detected after the laser excitationin addition to the shift at 668 nm indicating the formationof the P840⁺A₀^– state. The P840⁺A₀^– state decayedwith a t_1/e of 43 ns at 77 K and produced a triplet state p840^Tdue to the suppression of the forward electron transfer. Theseresults indicate the two different types of C670 species inthe RC complex; the one peaking at 670 nm functions as A⁰, whilethe other peaking at 668 nm shows the electrochromic shift,which presumably functions as the accessory pigment locatedin the close vicinity of P840. (Received May 17, 1999; Accepted July 14, 1999)     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

Limitations to net photosynthesis as affected by nitrogen status in jack pine (Pinus banksiana Lamb.) seedlings

Relative limitations of nitrogen (N) status on the processescontributing to photosynthetic rate (A) were investigated. Jackpine {Pinus banksiana Lamb.) seedlings from seeds grown in sandculture were supplied with four different N treatments for 6weeks, which resulted in a needle N content ranging from 50–85mmol m^–2 (14–32 mg g^–1 dry weight). Leaf gasexchange at varying CO₂ levels was measured and limitationson A₃₅₀ (A at ambient CO₂ level) caused by finite, limitingcarboxylation efficiency (c.e.), maximum A (A_max)and stomatalconductance were estimated from an analysis of the responseof A to internal CO₂ concentration. Although c.e. and A_max decreasedlinearly with the decline in needle N, the magnitudes of theirchanges relative to A₃₅₀ differed. A_max varied with A₃₅₀ andalways exceeded A₃₅₀ by 37–38% c.e., however, declinedfaster than A₃₅₀, as needle N level decreased. Consequently,relative limitation on A₃₅₀ caused by inefficient A_max remainedconstant, but limitations caused by c.e. increased by 10–15%at low N levels. In contrast, the limitation by stomatal conductancedeclined initially, but remained stable when N content droppedbelow 75 mmol m^–2. The results suggest: (1) a decreasein biochemical capacity, but not stomatal conductance, contributedto the reduction of A₃₅₀ induced by N-deficiency in jack pineseedlings; and (2) the capacity of carboxylation appeared tobe impaired more than that of electron transport and/or photophosphorylationand its reduction may be the major reason for the reductionin A₃₅₀. Key words: A–C_i analysis, carboxylation efficiency, electron transport, nitrogen deficiency, stomatal conductance     

The Osmotic Pressure of Concentrated Solutions of Polyethylene Glycol 6000, and its Variation with Temperature

The vapour pressures of aqueous solutions of polyethylene glycol6000 have been measured (by equilibration with sucrose solutions)up to the saturation point at 25 °C (1.45 g g^–1 water).The reduced-osmotic-pressure (/c), when plotted versus concentration(c), rapidly and linearly increased up to a concentration ofabout 0.8 g g^–1 (crossing the similar plot for sucrose).Above this concentration, the reduced-osmotic-pressure rosemore slowly, but still more rapidly than sucrose. The maximumosmotic pressure achieved at saturation was nearly 18 MPa. Usingthe virial equation: /c= RT/M + RTA₂c, the calculated secondvirial coefficient (A₂) for the linear part is 4.5 x 10^–3mol g^–1, a value slightly greater than most literaturevalues at 25 °C. Data are cited showing that A₂ varies linearlyfrom 5–6 x 10^x3 at 0 °C, to zero at 80–90 °C     

Synthesis of Citrulline and Arginine in Chlorella pyrenoidosa

The distribution of radioactivity in Chlorella during dark ¹⁴CO₂fixation was investigated either (a) in normal cells with andwithout added ammonium chloride, or (b) in nitrogen-starvedcells supplied with intermediates of the Krebs-Henseleit ureacycle. In the control experiments almost all the activity was presentin compounds of or associated with, the tricarboxylic acid cycle. The amino-acids citrulline and arginine became radioactive onlyin the presence of ammonia or ornithine where initially theycomprised 40–60 per cent. of the total activity, reactionsof the Krebs–Henseleit urea cycle being implicated intheir formation. No evidence could be found for a complete ureacycle. Unidentified compounds deriving their radioactivity fromthe C₄ carbon of citrulline and/or arginine were detected andformed up to 40 per cent. of the total ¹⁴CO₂ incorporated after25 min.     

A Comparison of the Growth, Photosynthesis, Stomatal Conductance and Water Use Efficiency of Moricandia and Brassica Species

When grown in pots and well-watered, the relative growth ratesof the above ground parts of two species of Moricandia (M. arvensis,an intermediate C₃–C₄ species, and M. moricandioides,a C₃ species) were inferior to those of two cultivated Brassicaspecies (B. campestris and B. napus). The Moricandia specieshad thicker leaves (greater d.wt per unit leaf area) with morechlorophyll than the Brassica species and had slightly greaterrates of photosynthesis per unit leaf area at an irradiance(400–700 nm) of 2000 µmol quanta m^–2 s ^–1.Leaves of M. arvensis, known to have a CO₂ compensation pointbetween that of C₃ and C₄ species, had a lower ratio of theintercellular to atmospheric partial pressure of CO₂ (C₁/C_a)and a greater instantaneous water use efficiency (WUE) thanthose of M. moricandioides and the Brassica species. Carbon isotope discrimination (     

Effects of surfactant distribution and ventilation strategies on efficacy of exogenous surfactant

The effectsof both surfactant distribution patterns and ventilation strategiesutilized after surfactant administration were assessed in lung-injuredadult rabbits. Animals received 50 mg/kg surfactant via intratrachealinstillation in volumes of either 4 or 2 ml/kg. A subset ofanimals from each treatment group was euthanized for evaluation of theexogenous surfactant distribution. The remaining animals wererandomized into one of three ventilatory groups: group1 [tidal volume(VT) of 10 ml/kg with 5 cmH₂O positive end-expiratorypressure (PEEP)]; group 2 (VT of 5 ml/kg with 5 cmH₂O PEEP); orgroup 3 (VT of 5 ml/kg with 9 cmH₂O PEEP). Animals wereventilated and monitored for 3 h. Distribution of the surfactant wasmore uniform when it was delivered in the 4 ml/kg volume. When thedistribution of surfactant was less uniform, arterial PO₂ values were greater ingroups 2 and3 compared with group1. Oxygenation differences among the differentventilation strategies were less marked in animals with the moreuniform distribution pattern of surfactant (4 ml/kg). In bothsurfactant treatment groups, a high mortality was observed with theventilation strategy used for group 3.We conclude that the distribution of exogenous surfactant affects theresponse to different ventilatory strategies in this model of acutelung injury.

     

Nutrient Solution pH Control using Dipolar Buffers in Studies of Trifolium repens L. Nitrogen Nutrition

In studies of Trifolium repens nitrogen nutrition, the controlof nutrient solution pH using dipolar buffers, was evaluatedin tube culture under sterile conditions. Five buffers; MES,ADA, ACES, BES and MOPS with pK₂s (20 °C) of 6.15, 6.60,6.90, 7.15 and 7.20 respectively, at a concentration of 2.0mol m^–3, were provided to inoculated Trifolium repensgrowing in nutrient solution containing 7.13 mol m^–3 nitrogenas (NH₄)₂SO₄. Initial pH of each solution was adjusted to theappropriate buffer pK₂ Two buffers, ADA and ACES completelyinhibited plant growth. The remaining buffers had little effectin limiting pH change, although plant dry matter was higherand nodule numbers lower in the presence of these buffers. MESand MOPS were supplied to nutrient solutions with and without7.13 mol m^–3 (NH₄)₂SO₄, at concentrations ranging from0–12 mol m^–3. MES at 9 mol m^–3 and 12 molm^–3 reduced growth of plants reliant on the symbiosisfor providing nitrogen. The provision of MES to plants providedwith NH₄⁺ significantly increased plant yield and reduced nodulenumber at all concentrations. MOPS did not affect plant yieldor nodule number. The use of dipolar buffers in legume nitrogennutrition studies is considered in terms of buffering capacity,and the side effects on plant growth and symbiotic development. Key words: Ammonium, Dipolar buffer, Nitrogen nutrition, pH control, Symbiosis, Trifolium repens