Further Observations on Light and Spore Discharge in Certain Pyrenomycetes

A ‘spore-clock’ for studying the hourly rate ofspore discharge over a 24-hour period is described. A numberof the experiments reported in this paper have involved theuse of this apparatus. In Sordaria fimicola there is a distinct positive light-dischargereaction in a dark-conditioned culture, the rate of spore dischargeincreasing steeply to a peak 2–3 hours after brief stimulationby bright light. Although darkening a light-conditioned cultureleads to an immediate decrease in the rate of discharge, thereis no evidence of a delayed negative dark-discharge reaction. In S. verruculosa with a 12-hours light: 12-hours dark dailyreëgime, more spores are discharged in the dark than inthe light periods if the intensity of illumination is low. Withhigher light intensity there is no significant difference betweenthe number of spores discharged in light and dark periods. Asin S. fimicola there is a positive light-discharge reaction,the interval between stimulus and maximum response being muchlonger (8–12 hours). When a dark-conditioned culture istransferred to light for 48 hours and then returned to darknessfor a further 48 hours it is apparent that not only is therea positive light-discharge reaction but also a negative dark-dischargeresponse. The ‘plateau’ level of discharge is essentiallythe same in light and darkness. It is confirmed that in Hypoxylon fuscum light inhibits discharge.     

Pathway of dark inorganic carbon fixation in two species of diatoms: influence of light regime and regulator factors on diel variations

Planktonic algae submitted to vertical mixing with a short periodicitycommute many times a day from low to high irradiance levels.To study the influence of this light periodicity, two diatoms,Skeletonema coslatum and Nitzschia turgiduloides, were cultivatedunder alternating conditions of 2 h light/2 h dark (2 h/2 h),simulating vertical mixing in the natural environment. Two otherlight regimes were used: continuous light (CL) and alternatecycles of 12 h light/12 h dark (12 h/12 h). Products synthesizedin the dark by S.costmum during 60 s incubation for 2 h/2 hculture or during 5 min for 12 h/12 h culture were determined.They were essentially sugars, malate, aspartate and glyceratefor 2 h/2 h cells and 12 h/12 h cells taken at the beginningof the light period. In contrast, 12 h/12 h cells taken duringthe darkness or in the middle of the light period and set inthe dark synthesized only amino acids. Our results corroborateprevious reports on dark CO₂ fixation via phosphoenolpyruvatecarboxykinase (PEPCKase, enzyme allowing the fixation of CO₂on PEP and the synthesis of amino acids) with involvement ofa substrate synthesized during the light period, but demonstratethat incorporation also occurs by the C-3 pathway (pathway responsiblefor the major CO₂ fixation in the light) in the very early stagesof the dark period. Another important result highlighted bythis study is the appreciable rate of dark fixation: on average6.7, 8.3 and 12.7% of photosynthesis at saturating photon fluxdensity for N.turgiduloides cultivated under 2 h/2 h, CL and12 h/12 h regime respectively and nearly 12% for S.costatumin the 2 h/2 h light regime. Variation of dark fixation wasinvestigated as a function of hour in the two species. Skeletonemacostatum cells submitted to the 2 h/2 h cycle show a constantrate of light-independent assimilation throughout the day. Bycontrast, both N.turgiduloides grown under the 12 h/12 h or2 h/2 h regime and S.costatum cultured under the 12 h/12 h cycleundergo fluctuations in the rate of dark fixation over the light/darkcycle. The mean dark fixation rate is controlled by the lengthof the photoperiod or the frequency of light fluctuations, dependingon species. We argue that this phenomenon must be taken intoconsideration in primary production calculations. Dependingon whether they are synthesized at the beginning or at the endof the light period, products are somewhat different and therate of fixation varies. This leads us to suggest that the pathwayof dark fixation may be regulated by at least two factors: amountof available substrate and enzyme (RuBPCase and PEPCKase) activityand/or amount.     

Novel Light-Dark Change of Proline Levels in Halophyte (Mesembryanthemum crystallinum L.) and Glycophytes (Hordeum vulgare L. and Triticum aestivum L.) Leaves and Roots under Salt Stress

Proline accumulation was determined in a facultative halophyte,Mesembryanthemum crystallinum and glycophytes, barley (Hordeumvulgare L.) and wheat (Triticum aestivum L.) Proline accumulationpreceded the shift of CAM in M. crystallinum and did not occurin the continuous darkness. The novel light-dark change of prolinelevel (high in the light and low in the dark) was observed inleaves of all three plants. Proline levels of shoots in barleyand wheat also showed the same light-dark change, suggestingthat proline accumulated in the leaves in the light was nottranslocated to other tissues in the dark period. These resultssuggest that proline has a bifunctional role in the acclimationto high salt stress; an osmoregulant role in the light, anda substrate for dark respiration to supply energy to compartmentationof ions into vacuole in the dark. ¹Present address: Kyoto Biological Res. Lab., Bio-Chiba Inc.Watsuka,Soraku, Kyoto, 619-12 Japan ²Present address: Kobayashi Pharmaceutical Co., Ltd. Doshomachi,Chuo-ku, Osaka, 541 Japan     

Modes of control by the circadian oscillator and the hourglass mechanism of the activities of cytoplasmic and chloroplast glyceraldehyde 3-phosphate dehydrogenases in Lemna gibba G3

Two types of clocks, i.e., the circadian oscillator and thehourglass mechanism, which under continuous light and darknessrespectively control the mutually inverse temporal changes inthe activities of Cyt-NAD-GPD and Chl-NADP-GPD of Lemna gibbaG3, were studied. Both clocks controlled the apparent Km values,not the Vmax values, of the GPD reactions for their substrateand coenzymes. A red light pulse inserted 3 hr after the onsetof the dark period eliminated the sigmoidal changes in darkness,but evoked rhythmical changes which otherwise did not occurin continuous darkness. Thus, the photosynthetic rhythm, ifpresent, would not sustain the GPD rhythms. This effect of ared light pulse was not nullified by a subsequent far red lightpulse. A far red light pulse given at the 3rd hour of an extendeddark period made conspicuous the sigmoidal changes in activityof GPDs in the dark period, and its effect was nullified bya subsequent red light pulse, suggesting that phytochrome isinvolved in the hourglass mechanism. (Received September 26, 1978; )     

Flowering in Pisum: the Effect of Light Quality on the Genotype If e Sn Hr

Far-red light, when given as a 16 h photoperiod extension, iamore effective than red light in reducing the flowering nodeof genotype Pisum. In contrast, when a 16 h dark period is interruptedby a 2 h light break red light is more effective than far-redlight. In addition, the stimulatory effect of a red interruptionis partially reversed by a subsequent period of far-red. However,a light interruption is not effective until over 12 h have elapsedsince the start of the previous photoperiod, regardless of whetherthe photoperiod was of 4 or 8 h duration. The results suggest that there are two light-dependent reactionscontrolling flowering in peas, one operating through the phytochromesystem with high levels of Pfr suppressing production of flowerinhibitor by the sn gene and a second requiring continuous illuminationwith wavelengths above 700 nm. The role of time measurementin the photoperiod response in peas is suggested to be filledby the proportion of time the Sn gene is effectively producinginhibitor. The photoperiod response in peas is not independentof temperature or plant age since the activity of gene Sn isalso varied by these factors.     

Environmental factors controlling the time of flower-opening in Pharbitis nil

Pharbitis nil, strain Violet, subjected to various photoperiods(24-hr cycle at 24?C) bloomed about 10 hr after light-off whenthe light period was 10 hr or longer, and about 20 hr afterlight-on when the light period was shorter. The higher the temperature(20–30?C) during the dark period, the later the time offlower-opening, with the temperature during the last half ofthe dark period having a stronger effect than that during thefirst half. In continuous dark or light, flower buds of Pharbitis openedabout every 24 hr at all temperatures tested between 20 and28?C, which suggests the participation of a circadian rhythmin determining the time of flower-opening. A light pulse given6–12 or 28–36 hr after the onset of the dark periodgreatly advanced the phase of this rhythm (8–10 hr). Phasedelay of this rhythm could not be obtained by light pulses givenat any time. (Received September 29, 1979; )     

Studies on the Photoreceptors for the Promotion and Inhibition of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy

Three-day-old etiolated seedlings of Pharbitis nil were exposedto red light for 10 min and sprayed with N⁶-benzyladenine beforetransfer to a 48-h inductive dark period, after which they weregrown under continuous white light. A second red irradiationpromoted flowering when given at the 5 and 24th hour of theinductive dark period but inhibited flowering at the 10 and15th hour. Far-red light inhibited flowering when given at anytime during the first 24 h of the dark period. Red/far-red reversibilitywas clearly observed at the 0, 5, 10 and 24th hour, but notat the 15th hour when both red and far-red lights completelyinhibited flowering. The action spectrum for the inhibition of flowering at the 15thhour of the inductive dark period had a sharply defined peakat 660 nm and closely resembled the absorption spectrum of theP_R form of phytochrome. The photoreceptors involved in thesephotoreactions are discussed. (Received June 10, 1983; Accepted July 6, 1983)     

In Vitro Studies of Floral Induction on Stem Apices of Cuscuta reflexa Roxb. --A Short-day Plant

Stem tips of Cuscuta reflexa, cultured on modified White's medium,were subjected to different light and dark conditions. The culturesflowered when maintained either in continuous darkness or exposedto 14 hours of daily dark period. Thus Cuscuta reflexa behavesas a typically short-day plant. The presence of 5 per cent.sucrose in the medium completely obviates the requirement forhigh-intensity light exposures, otherwise essential for SDP.It appears that the bud itself is sensitive to photoinduction.In spite of the presence of natural tissue-bridge between thehost and the parasite, provided by the haustorial connexions,there is no transportation of flower-forming substance fromone plant to another. The flowering periods of host and parasiteare independent of each other.     

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; )     

Growth and Dry-weight Distribution in Callistephus chinensis as Influenced by Lighting Treatment

Plants of two cultivars of Callistephus chinensis (Queen ofthe Market and Johannistag) were grown in 8 h of daylight perday with one of the following treatments given during the 16h dark period: (a) darkness—‘uninterrupted night’,(b) I h of light in the middle of the dark period—a ‘nightbreak’, (c) I min of light in every hour of the dark period—‘cycliclighting’, (d) light throughout—‘continuouslight’. The plants receiving uninterrupted dark periods remained compactand rosetted in habit with small leaves, while leaf expansion,stem extension, and flower initiation were promoted in all threeillumination treatments (b, c, d). Although these three treatmentsproduced similar increases in leaf area, continuous light wasthe most effective for the promotion of both stem growth andflower initiation while cyclic lighting was generally more effectivethan a I-h night break. Continuous light also caused more dry matter to be divertedto stems at any given vegetative dry weight and it was shownthat the stem weight ratio of both varieties was correlatedwith stem length.     

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.     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Regulation of PEP-Carboxylase by Biological Clock in a CAM Plant

The endogenous circadian rhythm in a crassulacean acid metabolism(CAM) plant Graptopetalum paraguayense was investigated. Phosphoenolpyruvatecarboxylase (PEP-C) takes two forms: the malate-sensitive dayform and the malate-insensitive night form. We monitored thestate of PEP-C by measuring the sensitivity to malate as a parameterof the circadian rhythm. We also measured vacuolar pH and malateconcentration, and contents of oxaloacetate, pyruvate and phosphoenolpyruvate(PEP). A free-running circadian oscillation was observed under continuousdim light (5 klux) after 12 h/12 h light/dark cycles at 20°C.The period of the rhythm was about 20 h. Under continuous light(18 klux), the rhythm was less clear but the length of the periodwas not affected. On the other hand, the rhythms of the vacuolarpH and the malate concentration were evident under the continuouslight, but were not clear under the continuous dim light. Therhythm disappeared in continuous darkness. The content of PEPchanged simultaneously with the transformation of PEP-C duringthe normal day-night cycles and under the continuous light,but stayed at a low level under the continuous dim light. Thisindicated that the transformation of PEP-C was not sufficientto maintain the rhythm in the carbon metabolism. Shift of the timing of the start or end of the dark period priorto the continuous illumination shifted the phase of the PEP-Crhythm without changing the period length significantly. At30°C, the rhythm of PEP-C was less clear, but the periodlength was not affected. These results suggest that the biological clock controls CO₂uptake and day-night CAM cycle through regulation of PEP-C transformation. (Received August 20, 1993; Accepted December 3, 1993)     

Regulation of photosynthetic carbon metabolism in synchronously growing Chlorella pyrenoidosa

The physical properties and photosynthetic metabolism of synchronizedcells of Chlorella pyrenoidosa are described. Cells, synchronizedby successive periods of light and dark, photosynthesized understeady-state conditions for 30 min with ¹⁴CO₂. Pool sizes ofmetabolic intermediate compounds, and rates of flow of carbonthrough these pools, were determined. Cell properties and metabolismwere studied for cells just divided, at three periods duringthe growing stage, at the time of maximum DNA synthesis, justprior to division (after continuous light, and after 5 hr darkness),and following division after continuous light for 37 hr. Changes in pool sizes and flow rates are correlated with relativeshifts between amino acid and protein synthesis, which is greatestduring the growing period and DNA-synthesizing stage, and sucrosesynthesis, which is greatest in the divided cells (after darkness)and pre-division cells (after darkness). The effects of thestage of cell growth and of a prior period of darkness can beseparated to some extent by these studies, and in some respectsare additive. Specific sites of metabolic regulation discerned in these experimentsinclude the following: 1) Ribulose diphosphate carboxylase,2) fructose diphosphatase and sedoheptulose diphosphatase, 3)the synthesis of sucrose, probably at the reaction between fructose-6-phosphateand uridine diphosphoglucose to give sucrose phosphate and uridinediphosphate, 4) amino acid synthesis, at the level of nitratereduction, and 5) amino acid synthesis, at the level of carbonflow from the photosynthetic carbon reduction cycle to aminoacid carbon skeletons. (Received October 31, 1969; )     

Studies on chloroplast development in Chlamydomonas reinhardtii I. Effect of brief illumination on chlorophyll synthesis

When dark grown cells of Chlamydomonas reinhardtii y-1 mutantwere exposed to continuous light, an immediate transformationof small amounts of protochlorophyll(ide), which had been presentin the dark grown cells, to chlorophyll was observed. Afterthis, there was a slow accumulation of chlorophyll lasting for2.5-3 hr before the start of exponential synthesis. Initialaccumulation of chlorophyll was distinctly slower at a highlight intensity (13,000 lux) than it was at moderate intensitiesof light (2,000–5,000 lux). However, the exponential synthesisof chlorophyll started after the same 2.5–3 hr of illumination. A brief pre-illumination of cells followed by incubation indarkness was effective in promoting chlorophyll synthesis undersubsequent continuous illumination at high, as well as moderatelight intensities. Pretreatment alleviated retardation of theinitial chlorophyll accumulation by light of high intensity.The promoting effect of preillumination on chlorophyll synthesiswas sufficient, even when a light impulse as short as 10 secwas given. However, the effect was dependent on length of thedark period after the short pre-illumination. The full extentof this effect was observed when the dark period was about 2.5–3hr long. Further dark incubation gradually decreased the effect. On the basis of these findings, it is assumed that a factor(s)responsible for promotion of chlorophyll (or chloroplast) synthesisin the process of greening of dark grown cells is produced duringthe dark period after a brief pre-illumination, and that thefactor is turned over at a relatively fast rate. The possiblenature of the presumed factor is discussed in relation to chloroplastdevelopment. ¹Present address: Department of Biology, Faculty of Science,Kobe University, Nada-ku, Kobe, Japan. (Received August 18, 1970; )     

Light-Induced Membrane Hyperpolarization and Adenine Nucleotide Levels in Perfused Characean Cells

This study examines the relationship between light-induced membranehyperpolarization and changes in adenine nucleotide levels intonoplast-free characean cells. When cells were perfused witha medium containing 1 mM ATP in the dark, the plasma membranedepolarized, the cytosolic ATP level decreased, and the ADPand AMP levels increased. Under light, the membrane hyperpolarized,the ATP level increased, and the ADP and AMP levels decreased.These changes in the adenine-nucleotide levels could partiallyexplain the membrane hyperpolarization. When cells were perfusedwith a medium containing an ATP-regenerating system consistingof phosphoenolpyruvate and pyruvate kinase, the membrane potentialremained in the hyperpolarized state, the ATP level remainedat a high level and no light-induced hyperpolarization was observed.The intracellular adenine nucleotide levels were also controlledby continuous perfusion. The membrane potential was determinedonly by the adenine nucleotide levels of perfusion media, irrespectiveof the light condition. Chloroplast-free Nitellopsis cells into which isolated Pisumchloroplasts were introduced also showed light-induced membranehyperpolarization. Pretreatment of chloroplasts with dicyclohexylcarbodiimide(DCCD) completely abolished the hyperpolarization with parallelinhibition of photophosphorylation. These results strongly suggestthat changes in adenine nucleotide levels caused by photophosphorylationare responsible for light-induced membrane hyperpolarizationin perfused cells. (Received August 17, 1985; Accepted December 13, 1985)     

Flower-promoting effect of some amino acids and amides in Lemna paucicostata 6746

Cultures of Lemna paucicostata 6746 were exposed to a single96-hr dark period followed by continuous illumination at 24?1?C.Flowering percentage increased to a maximum 3 days after theend of the dark period and then fell off to 0% on the 5th day.Among 20 amino acids and 2 amides tested, addition of asparagine,aspartate, glutamate, -alanine, glycine and serine clearly increasedthe flowering percentages and retarded the regression of floralbuds by 2–3 days. These substances given after the endof the long dark period were more effective than those givenduring the dark period, suggesting that they favored the flower-producingprocess following the inductive dark process. On the other hand,if the above amino acids or amide were applied under repeatedlight-dark cycles, they shortened the critical dark period by1–2 hr and almost completely nullified the light-breakeffect. They seem to promote the flower-inductive dark process,too. Glutamate, for instance, was effective even at 5 µM, whilethis amino acid is found in the plant body in large quantities.The mechanism of flower promotion by these amino acids and amideremains unknown. (Received June 3, 1976; )     

Studies on the Flowering Mechanism in Lemna

The dark reaction of the short day plant Lemna perpusilla was investigated. It was found that 3-phosphoglycerate and pyruvate (10^?6M) increased the flowering rate in the presence of nitrates. Pyruvate-2-¹⁴C was added to the culture solution during two hours of the dark reaction and ¹⁴C was incorporated into serine, aspartate and glutamate. It was postulated that pyruvate reacted with a nitrogen source forming an intermediate, possibly aspartate, which was further converted into serine. L. perpusilla failed to flower when the dark period was interrupted with red light and as a result endogenous serine accumulated in a high concentration. The dark reaction of L. perpusilla, in which serine was involved, required (1) oxygen, (2) ATP, (3) moderate temperature, and (4) an enzyme system.     

Elimination of the Far-Red-Induced Lag Phase of Chlorophyll a Accumulation by a Brief Light in Pharbitis nil Cotyledons

In dark-grown Pharbitis nil seedlings, far-red light (FR) irradiationof 48 h or less promotes Chl a accumulation in the first 2-hof a subsequent white light (WL) period, without a lag phaseof Chl a accumulation. However, continuous FR irradiation of72 h or more, causes the so-called FR-induced lag phase. A 5-minWL given 4 h before the onset of the continuous WL promotesChl a accumulation irrespective of the length of the precedingFR irradiation period, if a 4-h dark period is inserted betweenthe 5-min WL and continuous WL. This suggests that the effectof the brief WL is independent of and additive to the effectof the preceding FR irradiation, although the effect of theFR irradiation changes from promotive to inhibitory with anincrease in the irradiation period. Red light (R) is more active than blue light (B) in this brieflight effect. The R effect is reversed by subsequent exposureto FR when the period of the preceding FR irradiation is 24h, but not when it is 72 h. The relative effectiveness of Bto R increases after prolonged FR irradiation. (Received August 6, 1986; Accepted March 12, 1987)     

Effect of Light on Spore Discharge of Selected Basidiomycetes

Following entrainment of nine selected species of basidiomyceteswith alternating 12 h light and 12 h dark periods, circadianrhythms with nocturnal peaks of spore discharge were found topersist in continuous light or dark for periods of 4 –9d. However, when light and dark periods were reversed followingentrainment, Piptoporus belulinus. Panellus stipticus, Awiculariaauricula and Dacrymyces deliquescens made immediate readjustmentof their rhythms; Coriolus Dersicolor, Stereum hirsutum andClitocybe nebularis took 24 h to readjust with peaks of sporedischarge synchronized with the new dark time periods, whileGanoderma applanatum and Trametes betulina took 48 h to readjust.These reactions indicate exogenous rhythms Light effects, spore discharge, basidiomycetes