Effects of Blue Light on the Uptake of Ammonia and Nitrate by a Colorless Mutant of Chlorella

A colorless mutant of Chlorella kessleri, grown in darknessin a medium that contained nitrate and glucose, took up ammoniamore efficiently than nitrate. Irradiation with blue light inhibitedthe uptake of ammonia but, conversely, the uptake of nitratewas enhanced by blue light. These effects were not observedunder illumination with red or far-red light. The inhibitionby blue light of the uptake of ammonia was abolished in thepresence of nitrate. The charge compensation for the uptakeof ammonia was achieved by the immediate release of potassiumions and this release was followed by release of protons, therate of the latter process being strongly reduced by blue light. The effects of blue light on the uptake of ammonia and nitratein algal cells are discussed. (Received July 28, 1994; Accepted January 30, 1995)     

Wavelength effects on photosynthetic carbon metabolism in Chlorella

To study the wavelength-effect on photosynthetic carbon metabolism,¹⁴C-bicarbon-ate was added to Chlorella vulgaris 1 lh suspensionunder monochromatic blue (456 nm) and red (660 nm) light. Thelight intensities were so adjusted that the rates of ¹⁴CO₂ fixationunder blue and red light were practically equal. Analysis of¹⁴C-fixation products revealed that the rates of ¹⁴CO₂ incorporationinto sucrose and starch were greater under red light than underblue light, while blue light specifically enhanced ¹⁴CO₂ incorporationinto alanine, aspartate, glutamate, glutamine, malate, citrate,lipid fraction and alcohol-water insoluble non-carbohydratefraction. Pretreatment of the algal cells in phosphate mediumin the dark, which was essential for the blue light enhancementof PEP carboxylase activity, was not necessary to induce theabove wavelength effects. Superimposition of monochromatic bluelight at low intensity (450 erg.cm^–2.sec^–1) on thered light at saturating intensity caused a significant decreasein the rate of ¹⁴CO₂ incorporation into sucrose and increasein incorporation into alanine, lipid-fraction, aspartate andother related compounds, indicating that the path of carbonin photosynthesis is regulated by short wavelengdi light ofvery low intensity. Possible effects of wavelength regulationof photosynthetic carbon metabolism in algal cells are discussed. ¹ Part of this investigation was reported at the XII InternationalBotanical Congress, Leningrad, 1975 and the Japan-US CooperativeScience Seminar "Biological Solar Energy Conversion", Miami,1976. Requests for reprints should be addressed to S. Miyachi,Radioisotope Centre, University of Tokyo, Bunkyo-ku, Tokyo 113,Japan. ⁴ Present address: Department of Chemistry, Faculty of PharmaceuticalSciences, Teikyo Univ., Sagamiko, Kanagawa, Japan. (Received August 6, 1977; )     

Differential Effects of Methionine Sulfoximine on Light and Dark Uptake of Nitrate by Anabaena cylindrica

Methionine sulfoximine (MSX), a glutamine synthetase inhibitor,suppressed the inhibitory effect of ammonia on nitrate uptakeby Anabaena cells in both the light and dark. In the light,MSX did not inhibit nitrate uptake in the absence of ammonia,but under dark conditions, MSX above 2 µM inhibited nitrateuptake. Nitrite uptake, which is not affected by ammonia ineither the light or the dark, was inhibited by MSX in the darkbut not in the light. (Received October 3, 1984; Accepted April 22, 1985)     

Translocation Profiles of [11C] and [13N]-Labelled Metabolites after Assimilation of 11CO2 and [13N]-Labelled Ammonia Gas by Leaves of Helianthus annuus L. and Lupinus albus L.

Tracer amounts of atmospheric [¹³N]-Iabelled ammonia gas, wereabsorbed by leaves of Lupinus albus and Helianthus annuus inboth the light and the dark. Exogenous [¹³N]-ammonia was onlyabsorbed in the dark when the feeding occurred shortly aftera period of illumination and the tissue was not depleted ofits carbohydrate reserves (e.g. starch). Incorporation of the[¹³N]-ammonia appeared to occur via the leaf glutamine synthetase/glutamatesynthase (GS/GOGAT) cycle since 2.0 mol m^–3 MSX, an inhibitorof the GS reduced uptake in both the light and dark. Photosyntheticincorporation of ¹¹CO₂ was not affected by this treatment Therate of movement of [¹³N]-assimilates in the petiole of attachedleaves of Helianthus and Lupinus was similar to that of the¹¹Cl-photo assimilates. Export of both [¹³N] and [¹¹C]-Iabelledassimilates from the leaf and movement in the petiole in boththe light and the dark was inhibited by source leaf anoxia (i.e.nitrogen gas). Translocation was re-established at the samerate when the feed leaf was exposed to gas containing more than2% O₂ which permitted dark respiration to proceed. After aninitial feeding of either ¹¹CO₂ or [¹³N]-ammonia at ambient(21%) O₂ exposure of the source leaf to 2% O2, or 50% O² didnot alter the rates of translocation, indicating that changesin photosynthetic activity in the source leaf due to photorespiratoryactivity need not markedly alter, at least during the shortperiod, the loading and translocation of either [¹¹C ] or [¹³N]-labelledleaf products. Key words: Translocation, CO₂, NH₃, Leaves, Helianthus annuus, Lupinus albus     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

Suppressive effect of light on the formation of DNA and on the increase of deoxythymidine monophosphate kinase activity in Chlorella protothecoides

As previously demonstrated, chlorophyll-less cells of Chlorellaprotothecoides are obtained when the alga is grown in a mediumrich in glucose and poor in a nitrogen source (urea). When thesecells are incubated in a medium enriched with a nitrogen source,there occurs, besides greening of algal cells, an active formationof DNA followed by synchronous cellular division. The DNA formationand cellular division are markedly suppressed by light of acomparatively low intensity. Blue light is most effective andred light least effective in suppression. The effect of light on the level of dTMP kinase activity inthe algal cells was investigated in relation to the photoinhibitionof DNA formation. It was found that light suppresses the increaseof dTMP kinase activity in the algal cells which starts in advanceof active DNA synthesis, and that blue light has a strongersuppressive effect than red light. ¹Present address: Institute of Medical Science, University ofTokyo, Tokyo.     

Photoinhibition of Glucose Uptake in Chlorella

In colorless mutant cells of Chlorella vulgaris (M125), endogenousrespiration in the dark was not affected by 30-min preilluminationwith white light (9,000 mW?m^–2), while exogenous respirationof glucose or fructose was inhibited significantly by the sametreatment in air, but not under N₂. This light effect on exogenousrespiration was accompanied by an inhibition of hexose uptake. When autotrophically grown wild-type cells of Chlorella vulgaris(211-11h) were incubated in glucose medium with DCMU, lightalso greatly inhibited glucose uptake and growth. Blue lightwas very effective, while red light had only a slight effect.This photoinhibitory effect was also observed in algal cellsthat had been grown in a glucose-containing medium in the dark. Using SDS-gel electrophoresis, a new protein peak with a molecularweight of 35–40 kDa was detected in plasma membrane-richcell wall fractions when Chlorella vulgaris (211-11h) cellswere transferred to a glucose-containing medium. This peak disappearedafter the algal cells were returned to the glucose-free medium.These findings suggest that this protein includes the hexose-carrierprotein. Blue light significantly inhibited the formation ofthis protein during incubation in a glucose-containing medium. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)     

Effects of Light on Growth and Glucose Consumption in Colorless Chlorella Mutant Cells

Light inhibited the growth and glucose consumption of colorlessmutant cells of Chlorella vulgaris (# 125). Sugar consumptionwas also inhibited in a medium containing a hexose such as D-fructose,D-galactose and D-mannose. Blue light strongly inhibited growth and glucose consumptionbut red light only slightly affected them. Respiration was notinhibited by blue light. The inhibitions of growth and glucoseconsumption were saturated at light intensities as low as 800mW.m^–2 and continued in the dark for at least one dayafter brief illumination with white light. The half-maximumeffect was observed with 15 min of illumination in both casesand the action spectra for light-induced inhibitions of growthand glucose consumption were similar, both showing peaks at370, 457 and 640 nm. The role of light in the inhibitions of growth and glucose consumptionis discussed. (Received June 18, 1984; Accepted October 29, 1984)     

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.     

Some Biochemical Changes Related to Starch Breakdown Induced by Blue Light Illumination and by Addition of Ammonia to Chlorella Cells

Illumination with blue light enhanced the production of ammoniaby cells of C. vulgaris 11h, while no such effect was inducedby red light illumination. Addition of ammonia caused increasesin ATP levels and decreases in Pi and ADP levels. When 5 mMNH₄Cl was added to phosphorylase and amylase isolated from thecells of C. vulgaris 11h, their activities increased about 5–15%and 40–100%, respectively. (Received June 21, 1986; Accepted December 23, 1986)     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)     

The Photocontrol of Spore Germination in the Fern Ceratopteris richardii

This paper describes how different wavelengths of light regulatespore germination in the fern Ceratopteris richardii. This speciesdoes not exhibit any dark germination. Maximum photosensitivityof the spores is reached 7 to 10 d after imbibition. An increasein the red light fluence above the threshold fluence of 10¹⁶quanta.m^–2 leads to a corresponding increase in germination.In sequential irradiation experiments, farred light can reversethis red light-mediated germination to the level observed withthe far-red light control. Blue light fluences above 10²⁰ quanta.m^–2can also block the germination response to red light. Moreover,this antagonistic effect of blue light is not reversed by subsequentirradiation with red light. It is therefore concluded that phytochromeand a distinct blue light photoreceptor control C. richardiispore germination. These interpretations are entirely consistentwith the published literature on other fern genera. (Received November 28, 1986; Accepted April 6, 1987)     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells I. Chlorophyll Accumulation and Phototransformation of Protochlorophyll(ide) in Callus Cells under Blue and Red light

A marked accumulation of chlorophyll was observed in calluscells of Nicotiana glutinosa when they were grown under bluelight, while under strong red light no chlorophyll accumulated.This blue light effect saturated at an intensity of about 500mW.m^–2. The effects of white, blue and red light on the transformationof protochlorophyll (ide) (Pchl) accumulated in dark-grown calluscells were studied by following the changes in the intensityof fluorescence emitted by Pchl and different forms of chlorophyll(ide) (Chi). Pchl with a fluorescence maximum at 633 nm (absorptionmaximum: 630 nm) decreased slowly, concomitant with an increasein Chl having a fluorescence maximum at 677 nm (absorption maximum:675 nm), which was subsequently transformed, independently oflight, to Chi with a fluorescence maximum at 683 nm (absorptionmaximum: 680 nm). Both blue and red light of low intensitieswere effective for the phototransformation, while red light,but not blue light, of high intensities caused significant destructionof Pchl. An action spectrum for this photodestruction showedthat the maximum destruction took place at 630 nm. White lightof high intensities was effective for the photoreduction withonly slight destruction of Pchl, suggesting that blue lightcounteracts the destructive effect of red light. At low temperatures,however, blue light as well as red light of low intensitiescaused photodestruction of Pchl. It was inferred that blue lightenhances a certain step or steps involved in the productionof a reductant required for the photoreduction of Pchl to Chl. (Received July 3, 1981; Accepted November 11, 1981)     

Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea, Pisum sativum L.

Pathways of signal transduction of red and blue light-dependentacidification by leaf epidermal cells were studied using epidermalstrips of the Argenteum mutant of Pisum sativum. In these preparationsthe contribution of guard cells to the acidification is minimal.The hydroxypyridine nifedipine, a Ca²⁺-channel blocker, partlyinhibited the response to both blue and red light, while thephenylalkylamine, verapamil, a Ca²⁺-channel blocker that hasbeen shown in plant cells also to block K⁺-channels, causednearly complete inhibition. The Ca²⁺-channel activator S(–)BayK 8644 induced acidification when added in the dark and diminishedthe light-induced lowering of the extracellular pH. The Ca²⁺-ionophores,ionomycin and A23187 [GenBank] , also reduced the light response. Furthermore,the light-induced acidification was inhibited by the calmodulinantagonists W-7 and trifluoperazine, but not by W-5. These calmodulininhibitors completely inhibited the red light-induced acidification,but inhibited the response to blue light by only 60–70%.In general, inhibition by compounds affecting Ca-calmodulinsignalling was always stronger on the red light response thanthat on the blue light response (with the exception of verapamilthat blocked both the red and blue light responses equally well).This differential effect on red and blue light-induced responsesindicates a role for Ca²⁺-CaM signalling in both the red andblue light responses, while a second process, independent ofCa²⁺ is activated by blue light. Key words: Signal transduction, light-induced acidification, epidermal cells, pea     

Effects of Light on Degradation of Chlorophyll and Proteins during Senescence of Detached Rice Leaves

Regulatory effects of light on senescence of rice leaves wereinvestigated by measuring degradation of chlorophyll and proteinsin leaf segments which had been kept in the dark or under illuminationwith light of different intensities and colors. When leaveshad been left in total darkness for three days at 30°C,there was an initial long lag that lasted for one whole dayand then chlorophyll was rapidly degraded in the second andthird days. Breakdown of chlorophyll was strongly retarded bycontinuous illumination with white light of intensity as lowas 0.5 µmol photons m^–2 s^–1 but the effectof light decreased at intensities above 10 µmol photonsm^–2 s^–2. The initial lag and subsequent degradationof chlorophyll in the dark were little affected by illuminationwith red or far red light at the beginning of dark treatment.However, a brief illumination with red light at the end of thefirst and/or second day significantly suppressed degradationof chlorophyll during subsequent dark periods and the effectof red light was nullified by a short irradiation with far redlight. Thus, degradation of chlorophyll is regulated by phytochrome.Thylakoid membrane proteins and soluble proteins were also largelydegraded during three days in the dark. Degradation of membraneproteins such as the apoproteins of light-harvesting chlorophylla/b proteins of photosystem II and chlorophyll a-binding proteinsof reaction center complexes showed a long lag and was stronglysuppressed by illumination with weak white light. Thus, theloss of chlorophyll can be correlated with degradation of chlorophyll-carryingmembrane proteins. By contrast, light had only a weak protectingeffect on soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenaserapidly disappeared under illumination with weak white light.Thus, breakdown of thylakoid membrane and soluble proteins aredifferently regulated by light. Artifacts which would be introducedby detachment of leaves were also discussed. ¹ Present address: Department of Applied Biology, Faculty ofScience and Technology, Science University of Tokyo, Yamazaki,Noda-shi, Chiba, 278 Japan. ² Present address: Department of Life Science, Faculty of Science,Himeji Institute of Technology, Harima Science Park City, Hyogo,678-12 Japan.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta V. Photoinduction of 3-Hydroxy-3-Methyl Glutaryl Coenzyme A Reductase

The effect of light on the activity of 3-hydroxy-3-methylglutarylCoenzyme A (HMG-CoA) reductase in Rhodotorula minuta was studiedin cell-free extracts prepared from cells grown under variouslight conditions. HMG-CoA reductase activity in cells grown under continuous illuminationwas higher than that in cells grown in the dark, and dependedon the light intensity used during incubation. The relationshipbetween activity [A (nmol/mg-N/min)] and light intensity [I(erg/cm²/sec)] was expressed by the equation A=0.72 log I$0.80. Illumination at –1.5?C followed by dark incubation at26?C resulted in a rapid increase in HMG-CoA reductase activityimmediately after the beginning of incubation. This photoinducedHMG-CoA reductase activity was regulated by the light dose andfollowed the Roscoe-Bunsen reciprocity law. When cycloheximide was added immediately after the beginningof incubation in the dark, the increase in HMG-CoA reductaseactivity was completely inhibited. The inhibitory effect ofcycloheximide, however, gradually decreased with the delay ofthe addition. On the basis of these results we have postulated that the photoregulationof carotenogenesis in Rh. minuta results from the photoregulationof HMG-CoA reductase synthesis. (Received November 7, 1981; Accepted March 19, 1982)     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells II. Blue Light Effect on 5-Aminolevulinic Acid Formation

5-Aminolevulinic acid (ALA) accumulation in dark-grown tobaccocallus cells in the presence of levulinic acid (LA) was followedunder blue or red light or in continuous darkness. Significantformation of ALA continued in the dark. The protochlorophyll-(ide) (Pchl) content of dark-incubated cells remained low becauseof its turnover. We inferred that the feedback inhibition ofALA synthesis by Pchl would not occur in darkincubated calluscells. ALA formation was enhanced by blue light, and this effectreached saturation at an intensity of about 800 mW.m^–2.Neither weak nor strong red light affected ALA formation. Fullenhancement of ALA formation by blue light was attained afterfairly long continuous illumination of the callus cells. Thisblue lightenhanced activity of ALA synthesis declined very slowlyduring the subsequent dark incubation. The blue light enhancement of ALA formation was observed incallus cells supplied with sucrose over a wide range of concentrations.Pchl regeneration in carbon-starved callus cells, supplied withglutamate at various concentrations, was also markedly enhancedby blue light. Respiration of the callus cells was not enhancedby blue light. A possible role of blue light in regulating ALAformation in callus cells is discussed. ¹Dedicated to the late Professor Joji Ashida. (Received September 3, 1982; Accepted April 5, 1983)     

Involvement of the Accumulation of Glutamine in the Initiation of Adventitious Buds in Stem Segments of Torenia

Active meristematic divisions in stem segments of Torenia culturedin vitro can be induced in the epidermis by application of cytokininor the calcium ionophore A23187 [GenBank] , resulting in the differentiationof adventitious buds. Endogenous free glutamine accumulatedat a high concentration in the epidermal tissues during theearly stages of such cultures. The accumulation of glutaminewas caused by an increase in glutamine synthetase (GS) activity,and the increase of GS activity was suppressed by the applicationof some inhibitors of GS activity, mRNA synthesis, protein synthesis,or calmodulin. Incorporation of these inhibitors into the culturemedium also inhibited initiation of adventitious buds. The inhibitoryeffect of an inhibitor of GS, methionine sulfoximine (MSX),was apparent only at the very begining of the culture, and theeffect could be overcome by the simultaneous addition of glutamine.The inhibitory action of MSX on initiation of buds seemed tobe caused by an accumulation of ammonium ions. Reduction inlevels of NH₄NO₃ in or its elimination from the culture mediumstimulated the initiation of adventitious buds. Therefore, boththe accumulation of glutamine and the reduction in levels ofammonium ions seem to play a role in the initiation of adventitiousbuds in stem., segments of Torenia. ¹Present address: Faculty of Agriculture, University of Saga,Honjo-cho, Saga, Saga, 840 Japan. (Received October 3, 1988; Accepted March 9, 1989)     

BLUE LIGHT, PHYTOCHROME AND THE FLOWERING OF LEMNA PERPUSILLA 6746

Lemna perpusilla 6746 was grown on HUTNER'S medium with sucroseunder light schedules combining red, blue and far-red light.As shown earlier, brief red exposures added to a continuousblue schedule inhibit flowering although either schedule alonepermits it; hence red and blue act together in establishinga long day (flower inhibiting) condition. However, the red exposurerequired to inhibit flowering is greater with high intensitythan with low intensity continuous blue, suggesting in additiona blue-red antagonism. Blue light reverses the effects of abrief red exposure closing a blue or far-red main photoperiod,but it also reverses the effects of a brief far-red exposureclosing a red photoperiod. Thus, blue can act either like redor like far-red, depending on the situation. All effects ofblue light on the flowering of L. perpusilla 6746 are consistentwith the notion that it establishes a Pfr level intermediatebetween those established by red and far-red light; the postulationof an additional photoreaction to explain the effects of blueseems unnecessary. ¹Research carried out at Brookhaven National Laboratory underthe auspices of the U. S. Atomic Energy Commission.