Impact of light/dark regimen on growth rate,biomass formation and bacteriochlorophyll synthesis in Erythromicrobium hydrolyticum

The impact of illumination on specific growth rate, biomass formation, and synthesis of photopigment was studied in Erythromicrobium hydrolyticum, an obligately aerobic heterotrophic bacterium having the ability to synthesize bacteriochlorophyll a. In dark-grown continuous cultures the concentration of protein increased with increasing dilution rate, the concentration of bacteriochlorophyll a showed the opposite effect. At a dilution rate of 0.08 h^-1 (68% of _max in the dark) and S_R-acetate of 11.8 mM, the concentration of BChla of illuminated cultures in steady-state was 11–22 nM, compared to 230–241 nM in cultures incubated in darkness. No significant differences were observed in the concentration of protein. A shift from darkness to light conditions resulted in increased specific growth rates resulting in increased biomass formation, thus showing that light enhances growth by serving as an additional energy source. This phenomenon, however, was temporary because bacteriochlorophyll synthesis is inhibited by light. In contrast to incubation in continuous light or dark, incubation under light/dark regimen resulted in permanently enhanced biomass formation. In the dark periods, bacteriochlorophyll was synthesized at elevated rates (compared to constant darkness), thus compensating the inhibitory effect of light in the preceding period. It thus appears that the organism is well-adpated to life in environments with alternating light/dark conditions. The ecological relevance of the observations is discussed.Non-standard abbreviations BChla bacteriochlorophyll a - D dilution rate - spceific growth rate - K_s saturation constant - S_R concentration of limiting in inflowing medium of chemostat     

Valinomycin pulse-induced phosphorylation of ADP in dark anaerobic cells of the cyanobacteriumAnacystis nidulans

Addition of valinomycin to dark anaerobic suspensions ofAnacystis nidulans resulted in a transient hyperpolarization of the electrical potential across the cell membrane ( _CM) and seen from anilinonaphthalenesulfonate (ANS) fluorescence quenching and from distribution ratios of³H-tetraphenylphosphonium (TPP⁺) and¹⁴C-thiocyanate (SCN^–). At the same time a similar transient increase of intracellular ATP levels was observed, which was paralleled by decreasing ADP levels and eliminated by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the F₀F₁-ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD), and in the presence of K⁺ in the medium. Since the steady-state concentration of K⁺ in dark anaerobic cells was around 150 mM, it is concluded that a valinomycin-induced K⁺ diffusion potential across the cell membrane can serve as an energy source for ATP synthesis by a reversible H⁺-ATPase present in the membrane.     

Energetics of Active Phosphate Influx in Hydrodictyon africanum

The energy source for active phosphate influx in Hydrodictyonafricanum has been investigated using gas mixtures with andwithout O₂ and CO₂, light of various wavelengths, and metabolicinhibitors selective for respiratory or photosynthetic electrontransport and phosphorylation. It is concluded that, as in theother green algae studied, active phosphate transport requiresATP. In the dark this is supplied by oxidative phosphorylation;in the light the influx is much less sensitive to inhibitionof oxidative phosphorylation, and photophosphorylation (includingcyclic photophosphorylation) can act as energy source. Thissituation is more like that for active K influx (coupled toactive Na efflux) than to active Cl influx in H. africanum,except that the active dark influx is relatively greater forphosphate influx. The significance of these results for themechanism of regulation of light-stimulated ATP-requiring processes,and for the role of photosynthetic and oxidative phosphorylationin the energy metabolism of green cells, is discussed.     

Tip-localised H+-fluxes and the applicability of the acid-growth hypothesis to tip-growing cells: Control of chloronemal extension in Funaria hygrometrica by auxin and light

The role of tip-localised H⁺ secretion in regulating chloronemal tip growth in the moss Funaria hygrometrica Hedw. was investigated. pH was monitored with pH microelectrodes placed close to the cell surface while the rate of extension growth was manipulated by illumination and by the application of indole-3-acetic acid. Growth stimulations were accompanied by acidification of the external solution; this acidification was most pronounced at the growing tip. The timing and extent of acification external to the tip correlated well with the magnitude and time course of growth stimulations. The maintenance of both growth and H⁺ efflux under CO₂-free conditions indicated that neither photosynthetic nor respiratory CO₂ metabolism were involved. Artificially acidifying the nutrient solution rapidly but transiently stimulated elongation in both white light and darkness. Furthermore, the stimulation of elongation caused by white light was inhibited if the nutrient solution was buffered strongly near neutrality. We conclude that the acid growth hypothesis is applicable to tip growth in Funaria and that light and exogenous indole-3-acetic acid act at least in part by stimulating localised H⁺-ion efflux.Abbreviations D darkness - IAA indole-3-acetic acid - WL white light     

Effect of Inhibitors of Enzymatic DNA Methylation on the Formation of Reproductive Structures and Carotenoid Production in Neurospora crassa

The effect of inhibitors of DNA methylation on light-sensitive developmental stages of the filamentous fungus Neurospora crassawas studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3–300 M) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). After treatment of the mycelium with 5-azacytidine, the protoperithecia formation was accompanied by inversely proportional changes in the formation of conidia, both in the dark and after illumination. In the mycelium cultivated on the Vogel's medium, 5-azacytidine (up to 30 M) and methotrexate (up to 3 M) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.     

Pump-leak sodium fluxes in low salt corn roots

Summary The influx and efflux of sodium from 4-hr washed, low salt corn roots (Zea mays L.) has been studied for characterization of passive and active components. Initial Na⁺ content of the roots is very low, 2.25±0.4 mol/g fresh weight. Na⁺ influx in the presence of 0.2mm Ca²⁺ and 0.002 to 20mm K⁺ is passive (a leak) based upon Goldman-type models, being determined by Na⁺ and cell potential (). Na⁺ was not transported by the K⁺ carrier and influx was unaffected by 50 m dicyclohexylcarbodiimide (DCCD). Permeability of the cells to Na⁺ was of the same order asP _k.Efflux of Na⁺ was by an efficient and rapid active transport system (a pump), thus accounting for the failure of these roots to accumulate high levels of Na⁺. In short-term loading and efflux experiments, internal Na⁺ turnover had a half-time of about 5 min. Sodium efflux was unaffected by DCCD. Net H⁺ flux was zero in the presence of DCCD regardless of sodium efflux, indicating absence of Na⁺/H⁺ antiport. Efflux of Na⁺ was equally rapid into medium containing no Na⁺ and only 0.002mm K⁺. K⁺ influx accounted for less than 4% of Na⁺ efflux, prompting the hypothesis that the Na⁺ (or cation?) efflux pump is the second electrogenic system previously defined based upon electrophysiological measurements.     

Structural and Functional Rearrangements of Mesophyll as a Probable Basis for the Cytokinin-Dependent Assimilate Translocation in Detached Leaves

The effects of benzyladenine (BA) on the mesophyll functioning, such as osmotic potential (), the effect of the inhibitors of ⁺-ATPase on the influx of ¹⁴C-sucrose, the direction of carbon metabolism, and the rate of dark respiration, were followed in the detached leaves of pumpkin (Cucurbita pepo L.) and broad beans (Vicia faba L.). BA elevated and established a gradient of (_p) between the treated and untreated leaf regions. The inhibitors of H⁺-ATPase did not affect the BA-induced influx of ¹⁴C-sucrose. The changes were accompanied with the elevated synthesis of starch and other polymeric compounds and the diminished synthesis of the substances of relatively low molecular weight. The stimulation of dark respiration was short and inconsiderable. The author concludes that the BA-induced transport was a passive process related to a increase. Leaf expansion accompanied by the synthesis of high-molecular-weight substances essential for cell growth and by starch synthesis apparently increased the sink capacity of the BA-treated detached leaves. The diminished efflux from the leaf blade was probably related to a lowered level of the transportable carbon compounds restricting their entry into the phloem. The influx induction could result from the activation of growth and metabolic processes, the decline in the number of organic molecules per cell volume unit, and the development of _p between the source and sink leaf regions.     

Respirational activity of Chlorella fusca monitored by in vivo P-31 NMR

Energy metabolism during dark respiration of the green alga Chlorella fusca was investigated by ³¹P NMR spectroscopy. The kinetics of the transition from anaerobic to aerobic conditions (and vice versa) was followed with a temporal resolution of 16 s. This transition is accompanied by a shift of the cytoplasmic pH from 6.8 to 7.4, while the vacuolar pH remains constant. Simultaneously, an increase in the concentration of nucleoside-triphosphates and a decrease in the concentration of cytoplasmic orthophosphate take place, as well as the formation of mobile polyphosphates. The concentration of ATP and P _i reach steady-state levels within 30 s. Upon the reverse transition, from aerobic to anaerobic conditions, steady-state concentrations are obtained only after 3 min.     

Serum stimulation of sodium transport in human fibroblasts containing low and high levels of intracellular sodium

Summary The relationships between intracellular sodium content, sodium transport and serum effects were investigated in human fibroblasts. In the cells with low intracellular sodium (Na _iL ^/+ ;0.04 mol sodium/mg protein) serum stimulated the sodium-potassium pump as measured by ouabain-sensitive sodium efflux and rubidium influx and also exerted a transstimulation of ouabain-insensitive sodium transport resulting in net influx. In cells with high intracellular sodium (Na _iH ^/+ ;0.42 mol sodium/mg protein) all aspects of sodium transport were increased compared to Na _iL ^/+ cells. In these cells serum caused no change in sodium-potassium pump activity but significantly increased the ouabain-insensitive sodium fluxes resulting in net efflux. In Na _iL ^/+ cells, serum promoted net sodium influx through an amiloride-sensitive pathway that was undetectable in the basal state. In Na _iH ^/+ cells the serum-stimulated net efflux was amiloride sensitive but this pathway also contributed to a major portion of sodium transport in the basal state. This study demonstrated that sodium-potassium pump activity is directed by the supply of internal sodium and that serum can increase this supply by promoting net influx, and that serum-induced sodium transport can be modified by intracellular sodium content.     

Effects of UV-B on motility and photoorientation in the cyanobacterium,Phormidium uncinatum

UV-B irradiation has a detrimental effect on the survival of populations of the filamentous cyanobacterium, Phormidium uncinatum, at levels slightly higher than those currently measured at the surface of the earth. The organisms are not damaged or killed by UV-B radiation at 300 nm of 200 Wm^-2 for up to 20 h; but slightly increased levels of UV-B irradiation (2 h of 200 Wm^-2 at 300 nm) drastically impair motility, phototactic orientation and photophobic responses. These photosynthetic organisms require a narrow light intensity range for growth so that any decrease in their ability to actively search for and move into areas of favorable light conditions is bound to affect the survival of a population. The fluorescence yield of both phycobilins and chlorophyll is not altered even after 20 h of UV-B irradiation (200 Wm^-2 at 270 nm) indicating that UV-B at that dose does not affect the photosynthetic apparatus. The organisms are killed either by too bright intensities which bleach the photosynthetic pigments or by the lack of energy when they are unable to avoid moving into dark areas.     

Aspects of carbon metabolism in Chloroflexus

When fluoroacetate was added to aerobic, washed cells of Chloroflexus, O₂ uptake was strongly inhibited and citrate accumulated. Under anaerobic conditions in the light, fluoroacetate inhibited CO₂ uptake and caused citrate accumulation. The results are taken as evidence for the operation of a tricarboxylic acid cycle in Chloroflexus both under aerobic conditions in the dark and anaerobically in the light. 2. Organic compounds are assimilated into the storage materials polyglucose and poly--hydroxybutyric acid by washed cells of Chloroflexus. The type of storage product formed from acetate depends upon the availability of reducing power. 3. Low activities of the key enzymes of the reductive pentose phosphate cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase were detected in cell free extracts of photoheterotrophically grown Chloroflexus.Abbreviations RuBP Ribulose-1,5-bisphosphate - TCA tricarboxylic acid - PHB poly--hydroxybutyric acid     

Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g^-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g^-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (= pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no pH could be measured in the incubation medium, these results can be best explained by a H⁺-hexose cotransport mechanism powered by active H⁺ extrusion at the plasmalemma.Abbreviations LD longday - SD shortday - pd membrane electropotential difference - pd maximum membrane potential depolarization - L light - D dark - FW fresh weight - d days of culture of Lemna gibba - 1X perfusing solution without sugar, see methods     

Characterization of calcium fluxes across the envelope of intact spinach chloroplasts

Calcium fluxes across the envelope of intact spinach chloroplasts (Spinacia oleracea L.) in the light and in the dark were investigated using the metallochromic indicator arsenazo III. Light induces Ca²⁺ influx into chloroplasts. The action spectrum of light-induced Ca²⁺ influx and the inhibitory effect of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) indicate an involement of photosynthetic electron transport in this process. The driving force for light-induced Ca²⁺ influx is most likely a change in the membrane potential component of the proton motive force. This was demonstrated by the use of agents modifying the membrane potential (lipophilic cations, ionophores, different KCl concentrations). The activation energy of the observed Ca²⁺ influx is about 92 kJ mol^-1. Verapamil and nifedipine, two Ca²⁺-channel blockers, have no inhibitory effect on light-induced Ca²⁺ influx, but enhance ferricyanide-dependent oxygen evolution. Inhibition of Ca²⁺ influx by ruthenium red reduces the light-dependent decrease in stromal NAD⁺ level.Abbreviations and symbols Chl chlorophyll - DCMU 3-(3',4'-dichlorophenyl)-1,1-dimethylurea - FCCP earbonyl cyanide p-trifluoromethoxyphenylhydrazone - PGA 3-phosphoglyceric acid - ABA⁺ tetrabutylammonium chloride - TPP⁺ tetraphenylphosphonium chloride - E membrane potential     

DCCD induced sodium uptake by Anacystis nidulans

The Na level inside cells of Anacystis nidulans is lower than in the external medium reflecting an effective Na extrusion. Na efflux is an active process and is driven by a Na⁺/H⁺-antiport system. The necessary H⁺-gradient is generated by a proton translocating ATPase in the plasmalemma. This ATPase (electrogenic proton pump) also produces the membrane potential (about -110 mV) responsible for K accumulation. N,N-dicyclohexylcarbodiimide (DCCD) inhibits the ATPase and the H⁺-gradient completely, but the membrane potential is only reduced (<-70 mV), since K efflux initiated by DCCD maintains the potential partly by diffusion potential.With DCCD, active Na efflux is inhibited thus revealing Na uptake and leading by equilibration to the membrane potential to a 5–20 fold accumulation. Na uptake depends on the DCCD concentration with an optimum at (1–2)×10^-4 M DCCD. Pretreatment with DCCD for a few minutes followed by replacement of the medium suffices to induce Na uptake.DCCD induced Na influx is about 5 times faster in light than in darkness, and the steady state is reached much earlier in light; a 5 fold increase by light was also found for Rb uptake with untreated cells. Valinomycin stimulates the influx of Rb to about the same rate in light and dark. Therefore light may unspecifically increase the permeability of the plasma-lemma probably via the ATP level. Similarly to DCCD also 3×10^-3 M N-ethylmaleimide induces Na uptake.Abbreviations Used DCCD N,N-dicyclohexylcarbodiimide - NEM N-ethylmaleimide - CCCP carbonylcyanide m-chlorophenylhydrazone - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Effect of light on vanillin precursors formation by in vitro cultures of Vanilla planifolia

A cluster culture of Vanilla planifolia Andr. has been established from shoots. It has been maintained in the dark. Various light conditions had little effect on its growth. However, the light conditions did affect the production in both quantity and quality of compounds associated with the vanillin pathway, particularly the 4-hydroxy-3-methoxybenzyl alcohol.Abbreviations NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)-purine - BA 6-benzylaminopurine     

Nitrogen isotope discrimination in white spruce fed with low concentrations of ammonium and nitrate

Differences in ¹⁵N among ten white spruce [Picea glauca (Moench) Voss] families were examined in hydroponic experiments testing (1) three N sources [100 M N as (i) NH₄⁺, (ii) NO₃^– or (iii) NH₄NO₃] and (2) two supply regimes [200 M NH₄⁺ (i) maintained steadily or (ii) recurrently drawn-down]. In the N-source experiment, the NH₄⁺ treatment resulted in superior growth and lower C/N ratios. Whole plant ¹⁵N was higher on NH₄⁺ and NH₄NO₃, reflecting higher NH₄⁺ removal rates from the media. Families expressed differences in biomass, C/N, ¹⁵N and ¹³C. Family ¹⁵N and ¹³C were positively correlated in the NH₄NO₃ treatment and the steady-state regime. Supply regime did not affect total biomass, but higher root/shoot ratios implied N was more limiting under the draw-down regime. Family rank changed with supply regime, but not with N source. Analysis of media isotope enrichment during substrate depletion revealed relationships between net discrimination and external N concentration. Discrimination against ¹⁵NH₄⁺ was about twice that of ¹⁵NO₃^–. A simple model relating isotope discrimination to relative rates of ion efflux and influx predicted efflux/influx ratios consistent with published values for white spruce. We propose that genetic differences in discrimination are caused by different demands on assimilation and in uptake capacity which interact, influencing the balance between N influx and efflux.     

Phototactic responses in Haematococcus lacustris and its modification by light intensity and the carotenoid biosynthesis inhibitor Norflurazon

At fluence rates below 45 W· m^-2 cells of the flagellate stage of Haematococcus lacustris react only positively phototactically with a rather high degree of orientation (indicated by r values up to 0.66 with the Rayleigh test). The directedness of orientation decreases with decreasing irradiance. The degree of directedness of the phototactic response depends on the intensity of preirradiation: Low light intensity applied after strong light application results in a dark reaction (low r values), low light given after darkness stimulates a rather high degree of directedness of positive phototaxis. Weak blue light (=483 nm; 0.4 W · m^-2) stimulates positive phototactic response, whereas comparable red light (=658 nm; 0.5 W · m^-2) does not.Cells which were grown in a medium containing 10^-4 M Norflurazon (effective in inhibition of carotenoid biosynthesis) although maintaining motility completely lose the ability to react positively phototactically. The possible role of carotenoids in the phototactic orientation is discussed.     

Wirkung von hemmstoffen der DNS-, RNS- und proteinsynthese auf wachstum und antheridienbildung in prothallien von Anemia phyllitidis L.

Summary A number of inhibitors of DNA-, RNA-, and protein synthesis were applied to prothallia of Anemia phyllitidis, and their effects were investigated with regard to cell division and inhibition of antheridium formation (after induction by gibberellin A₃). All compounds tested cause a significant inhibition of growth accompanied by teratologies typical for each inhibitor. Only 5-bromouracil proved to be inactive, even at a concentration of 8×10^-4m.None of the inhibitors blocked the induction of the biplanar growth form in continuous light.With the exception of 5-bromodeoxyuridine and 5-iododeoxyuridine all the antimetabolites investigated cause a significant lag in antheridium formation. The result of an analysis of this inhibition on the basis of the critical cell number (Schraudolf, 1966a) demonstrates that this time lag is clearly a direct consequence of the retardation of cell division caused by the inhibitors. There is no inhibition of the induction process proper. So therefore it appears highly improbable that antheridium induction in Anemia by gibberellins is based on a specific gene activation in the sense of Jacob and Monod.The results presented in this paper demonstrate that the production of a time lag in the realisation of a process of differentiation or morphogenesis is not sufficient evidence to permit definite conclusions to be drawn about the basic mechanism of induction; the effect of inhibition of cell division caused by the inhibitors must be excluded or at least taken in to account.The possible function of gibberellins as realisators of already present but inactive m-RNA is discussed.5-Bromo- and 5-iododeoxyuridine do not cause a time lag in antheridium formation; both inhibitors, however, cause a deformation of the cell pattern of the antheridia. The degree of this deformation depends on inhibitor concentration as well as on exposure time. Probably a competition exists between these halogensubstituted pyrimidines and thymidine for incorporation into DNA.     

Paramylon (β-1,3-glucan) content in wild type and WZSL mutant of Euglena gracilis. Effects of growth conditions

The effects of growth conditions on paramylon (a -1,3-glucanreserve carbohydrate) content were examined in the photosyntheticwild-type and a spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. This carbohydrate is known toshow interesting applications in human and veterinary medicine, asimmunostimulant and immunopotentiator. For both strains, the synthesisof reserve depends mainly on the growth conditions, i.e. light or dark. Thehighest amount of paramylon is accumulated by the mutant (90% dryweight) under dark conditions and with glucose as the carbon source. Thesefindings are discussed in terms of feasibility of exploitation of both strainsas an alternative source of -glucan, and of the importance of thechloroplast compartment in the synthesis of this compound.     

Nitrogen-limited continuous culture ofRhodopseudomonas capsulata growing photosynthetically or heterotrophically under low oxygen tensions

A continuous culture apparatus is described, which allows cultivation of photosynthetic bacteria anaerobically in the light and semiaerobically in the dark at constant oxygen tensions. The growth-parameters 1. substrate concentration at half maximum growth rate (K _s) and 2. yield (Y) forRhodopseudomonas capsulata are calculated.An automatic control system of the oxygen partial pressure in the culture medium is elaborated. It is shown, that the discontinuous regulation with a control unit in connection with two magnetic valves, which give short pulses of either oxygen-free gas or gas mixed with oxygen, is an economic, practicable and reliable method.The yield coefficientY during growth limited by ammonium sulfate is variable due to the synthesis of nitrogen independent metabolites, such as poly--hydroxybutyrate. The storage of poly--hydroxybutyrate in continuous culture is a function of both the actual concentration of ammonia and of theC/N ratio. At very low growth rates (1/6 µ _m ) the poly--hydroxybutyrate content increases amounting to 33% of the dry weight.In semiaerobically dark grown cells (pO₂: 5 mm Hg) the protein and bacteriochlorophyll content increased definitely on dry weight basis with increasing growth rates. In contrast, in anaerobic light cultures only a small increase of the bacteriochlorophyll level but no change of the protein content per dry weight of cells was observed at increasing growth rates.