Diurnal rhythm of uridine incorporation into RNA regulated by two light-perceiving systems in a long-day duckweed, Lemna gibba G3

A long-day duckweed, Lemna gibba G3, was found to be controlledby two lightperceiving systems; a system perceiving a prolonged,high-intensity white light and the phytochrome system, withrespect to the incorporation of radioactive uridine into RNA.When the duckweed was exposed to short or long days, the uridineincorporating activity into RNA changed diurnally reaching itshighest level at 18 hr and its lowest one at 6 hr after thebeginning of a light period. The level of maximum activity rosein proportion to an increase in the length of the light periodup to 12 hr or in light intensity up to 3000 ergs/cm²sec. Thefar-red light termination of the light period resulted in adecrease in uridine incorporation, the extent of which was constantirrespective of the length of the light period. The uridine incorporating activity changed diurnally when theduckweed was exposed to continuous light. The period lengthof the rhythm was circadian and was constant over a temperaturerange of 16° to 30°C. (Received September 1, 1975; )     

Endogenous light-on rhythm in respiration of a long-day duckweed, Lemna gibba G3

1. The rate of O₂-uptake of Lemna gibba G₃ changed with a dampeddiurnal rhythm under continuous illumination given after shortdays. The rhythm was started by a light-on stimulus with a 6hr lag period and is thought to be under the control of a biologicalclock. 2. The 6 hr lag period was replaceable with a 6 hr dark periodinterrupted twice (at 0 and 3 hr) by a brief illumination withred light. The effect of red light was removed by immediateexposure to far-red light. This effect of far-red was reversedby subsequent red light. The 6 hr lag may involve a phytochrome-mediatedreaction which may be preparatory to the induction of this rhythm. (Received December 13, 1969; )     

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; )     

THE RHYTHMICAL CHANGE IN SENSITIVITY OF A LONG-DAY DUCKWEED, LEMNA GIBBA G3, TO DARK-BREAK

1. Effect of varied lengths of darkness given before continuousillumination, and that of dark-break of continuous light asa function of the time of its application, on the flower formationin a long-day duckweed, Lemna gibba G3, were studied. The results obtained suggested a rhythmic change in sensitivityto darkness, i.e., a cycle of 36 hr-period consisting of 12hr of sensitivity and the following 24 hr of insensitivity.The inhibition by darkness (12–36 hr) given before thestart of, or by dark-break (12, 24 hr) inserted in, the inductionperiod involved an extension of the induction period, but nota slow-down of the rate of flower formation. The dark-breakgiven after the induction period, however, suppressed the rateof flower production in proportion to the length of the darkness. 2. The inhibition of flowering by darkness given in the darksensitivephase was cancelled by a relatively brief light period insertedin the darkness. 3. Relation between the rhythm and the length of induction periodwas discussed. (Received August 13, 1965; )     

A Nitrate Reductase-Inactivating Factor from Barley (Hordeum distichum L.) Leaves

A nitrate reductase-inactivating factor (NR-IAF) was detectedin a crude extract from 8-day-old barley (Hordeum distichumL. cv. Daisen-gold) leaves by chromatofocusing. The factor seemedto be a proteolytic enzyme with a cysteine residue at its activesite because 1) it was thermo-labile, and trypsin treatmentcaused loss of activity; 2) p-chloromercuribenzoic acid andiodoacetamide inhibited its activity; 3) leupeptin, an inhibitorof trypsin-like enzymes, also inhibited its activity; and 4)proteolytic activity toward azocasein was detected for the factorpreparation. The factor did not affect the activities of nitritereductase, glutamate dehydrogenase and xanthine oxidase. (Received March 22, 1983; Accepted July 30, 1983)     

Ethylene Production in Sweet Potato Root Tissue Infected by Ceratocystis fimbriata

The rate of ethylene production by sweet potato (Ipomoea batatasLam. cv. Norin No. 1) root tissue infected with Ceratocystisfimbriata Ell. & Halst. increased markedly during incubationat 29?C under high relative humidity. During incubation thefungus progressively invaded root tissue. The rate of ethyleneproduction reached a peak two days after inoculation when thebrowning region that contained the penetrating mycelia had expandedinward about 0.3 mm from the surface, followed by a declinein ethylene production. Apparently, the 1-aminocyclopropane-1-carboxylicacid (ACC) synthase activity was not high enough, and the amountof ACC in the infected tissue was too low to account for thehigh rate of ethylene production throughout the incubation period.Ethylene production by the infected tissue showed scarcely anyinhibition by amino-ethoxyvinylglycine, a specific inhibitorof ACC synthase. These findings suggest that the pathway ofethylene biosynthesis that operates in infected sweet potatoroot tissue may differ from the methionine pathway in whichACC serves as an intermediate. (Received March 24, 1984; Accepted June 27, 1984)     

Effects of light on fruit-body formation in a basidiomycete, Coprinus macrorhizus

A basidiomycete, Coprinus macrorhizus produced only vegetativemycelia, when cultured under continuous darkness. Under continuouswhite light, visible tiny primordia formed on the 6th day afterinoculation, followed by normal development to fruit-bodies.Spores disseminated on the 11th day. However, when cultureswhere transferred to continuous darkness after the formationof primordia, rudimentary pilei with slender stalks formed.Abundant hairy hyphae were produced along the entire surfaceof the slender stalks. Thus, light was required-for both theinitiation and development of the fungal fruit-body. A fairy ring of primordia formed along the narrow region ofmycelia formed just prior to the exposure to light, provideda colony was pre-grown for more than 4 days in the dark, thenexposed to light for 24 hr or longer. Exposure to light for at least 48 hr during the period between24th and 96th hr after primordium initiation was required fornormal maturation of the fruit-body. This 48 hr light periodneeded for maturation of the fruit-body could be substitutedby two, 2 hr light periods given at the beginning and the endof the 48 hr period. We have tentatively concluded that lightis required at two definite stages for fruit-body developmentafter formation of the primordium. Effective wavelengths for both the initiation and developmentof fruit-bodies were in the near ultraviolet and blue regions. (Received July 24, 1972; )     

Activation of phospholipase D by calmodulin antagonists and mastoparan in carnation petals

An in vivo assay for phospholipase D (PLD; EC 3,1,4,4) activity,based on its transphosphatidylation property, is described indetail and was used to study putative post-translational regulationmechanisms of PLD activity in carnation (Dianthus caryophyllusL.) petals. A variety of agents was applied to petal discs.The calmodulin (CaM) antagonists propranolol, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide(W7) and N-(6-aminohexyl)-1-naphthalenesulphonamide (W5), stimulatedPLD activity in a dose-dependent manner. EGTA partially inhibitedthe stimulation by the CaM antagonists. Erythrosin B, an inhibitorof CaM-dependent P-type Ca^z+-ATPases, slightly stimulated PLDactivity. The results suggest that part of the stimulation ofPLD activity by CaM antagonists is due to an increased intracellularCa²⁺-concentration, PLD activity was stimulated by mastoparanin a dose- and time-dependent manner. The signal-like activationkinetics suggests that mastoparan activates PLD (in)directlyvia a G protein. Key words: Phospholipase D, CaM antagonists, mastoparan, Dianthus, calcium     

Effect of light on the initiation of pileus formation in a basidiomycete, Favolus arcularius

Pileus formation in Favolus arcularius is induced by light,but no photoinduction occurred in young epileate stipes. Thestipes usually had to attain a length of about 5 mm to be photosensitive.Synchronous pileus formation could be induced by exposure tolight using epileate stipes which had been preincubated in darknessfor 48 to 72 hr. The pileus primordium formed about 24 hr afterthe start of illumination, however, continuous illuminationwas not necessary to produce this effect. A dark period givenbetween 1 and 8 hr after the start of illumination did not retardpileus formation. The photoinduction of pileus formation involvedtwo light-requiring processes, one occurring during the firsthour (the first light process) and the other from the 8th tothe 24th hr (the second light process). The photoresponse inthe first light process was saturated with 5 lux of light, buta light intensity below 1 lux was essentially ineffective. Onthe other hand, the reaction in the second light process couldbe started by less than 2 lux, and was accelerated by increasingthe light intensities up to about 150 lux. Further increasesin light intensity did not improve any significant effect. (Received April 30, 1974; )     

Photoreversibility of flower initiation in Lemna perpusilla as affected by the light quality of the main light period

Reversible floral responses of Lemna perpusilla to red and far-redlights appeared only at the beginning of the inductive darkperiod when the 8 hr photoperiod consisted of white or red light.When blue or far-red light was given during the 8 hr photoperiod,the far-red given at the beginning of the dark period scarcelyinhibited flowering; red/far-red reversibility newly appearedat the middle of the dark period. This indicates that the photoregulationsystem in the flowering of L. perpusilla can be converted fromthe Pharbitis type to the Xanthium type by changing the lightquality of the main photoperiod from white or red to blue orto far-red, which is known to be effective for the so-calledhigh-energy photoreaction of photomorphogenesis. (Received July 2, 1975; )     

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; )     

Diurnal change of light-dependent uridine incorporation into RNA in a long-day duckweed, Lemna gibba G3

Light-dependent incorporation during subjective day and nightof radioactive uridine into RNA of a long-day duckweed, Lemmagibba G3, was examined. When the dark treatment was startedfrom the subjective night phase, the activity of uridine incorporationdropped approximately by half only after the very subjectivenight phase had passed or with the commencement of the subsequentsubjective day phase. However, when the dark treatment was startedfrom the subjective day phase, the incorporating activity promptlybegan to decrease and the inhibition increased with the lengthof the dark period until a final steady level (also at ca. 50%of the initial level) was reached after 24 hr of darkness. Thesetwo phases of different light sensitivities recurred daily undercontrol of the physiological clock and the rhythm was resetby a light-on signal. The lowered incorporating activity dueto the darkened day phase was recovered completely by a 12-hror even 15-min white light period perturbing the succeedingnight phase. That the incorporation of uridine in every RNAspecies, especially in chloroplast ribosomal RNA, was loweredby dark treatment of the day and night phases, was disclosedby MAK column chromatography and acrylamide gel electrophoresis. (Received August 21, 1974; )     

The relationship between red and far-red light on flowering of the long-day plant, Lemna gibba

Lemna gibba, a long-day duckweed, can be induced to flower whenthe 10 hr white photoperiod is extended with red or far-redlight. The 10 hr red photoperiod is also effective in inducingflowering when followed by a far-red extension, but a red extensionis ineffective. When 2 hr of far-red light are given immediately after the 10hr red photoperiod, the following red as well as the far-redextension can induce flowering, indicating that the 2 hr far-redlight plays an important role as a starting factor for induction.This red or far-red extension is effectively replaced by a redbreak given at a proper time in the darkness which follows the2 hr far-red light as the starting factor. The effect of thered break in not cancelled by subsequent exposure to far-red,which synergistically promotes flowering. However, a red break given immediately after a proper periodof far-red extension further promotes flowering. The phase sensitiveto the red break coincides with that sensitive to the red breakgiven in darkness. The effect of the red break is reversed bysubsequent exposure to far-red, contrary to the effect of thered break in darkness. Using these results, relation between red and far-red lighton flowering in L. gibba is discussed. (Received July 17, 1971; )     

RNA metabolism in apices of Pharbitis nil daring floral induction

RNA metabolism was studied in apices of Pharbitis nil duringand after floral induction. In continuous light ³H-uridine accumulatedin RNA at a constant rate over an 18 hr period. In darkness,however, the rate of accumulation of label into RNA was constantuntil the 10th hour at which time a rapid burst of accumulationoccurred, peaking at the 14th hour of darkness and followedby a net loss of label. The RNA involved in this burst is probablymRNA due to its size and poly(A) content. This phenomenon doesnot seem to be associated with floral induction, since the siteof perception is the apex, and it also occurs under conditionswhere floral initiation is inhibited by a brief light interruptionof the dark period. Immediately after floral induction by a16-hr dark period the rate of RNA synthesis was suppressed about14%. This suppression lasts for about 12 hr and was followedby a twofold increase in the rate of RNA synthesis, comparedto non-induced apices, at 64 hr after the beginning of the inductivedark period. These post-induction changes were found to occurin all RNA fractions. ¹Present address: Department of Radiation Biology and Biophysics,University of Rochester School of Medicine and Dentistry, Rochester,N.Y. 14642, U.S.A. (Received March 15, 1976; )     

Effects of Low Temperature, Light and O2 on Chilling-sensitive and -resistant Strains in Chlorella ellipsoidea

A low-temperature sensitive strain, Chlorella ellipsoidea Gerneck(IAM C-102), lost its chilling sensitivity during preservation.Cells of the original strain (low-temperature sensitive) andthe variant (low-temperature resistant) were both synchronouslygrown under a 14-hr light-10-hr dark regime. In the originalstrain, cells at the D-L stage (transient phase) were most sensitiveto a low temperature, whereas the variant cells were not damagedat any stage. During low-temperature treatment, the viability of D-L cellsin the sensitive strain decreased after a lag period of 1 hr.The O₂-uptake activity (respiration) showed the same behavioras the viability, whereas the O₂-evolution activity (photosynthesis)decreased from the start of chilling. In the resistant strain,only O₂ evolution decreased. The decreased activity was restoredwhen the chilled cells were incubated at 25°C. This restorationwas inhibited by oligomycin. Lowering the light intensity or eliminating O2 diminished thechilling injury of the sensitive strain. The results indicatethat the chilling injury of Chlorella results from the combinedeffects of low temperature, light and O₂. (Received September 26, 1980; Accepted March 23, 1981)     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )     

Maintenance of high Pfr-level in the dark period in relation to flowering in Lemna gibba G3

The relative Pfr-level in a long-day duckweed, Lemna gibba G3,was estimated by the null response method. The null % R value(% R in a R/FR-mixture that provides a null flowering response.This value was assumed to indicate the endogenous Pfr-levelof the duckweed.) remained high during the initial hours ofthe 15 hr nyctoperiod then decreased rapidly, if a 12 or 33hr photoperiod preceded the nyctoperiod. The null % R valuedropped immediately after the start of the 15 hr nyctoperiodsubsequent to a 1 or 24 hr photoperiod. Thus, the duration ofthe maintenance of a high Pfr-level changed rhythmically dependingon the length of the preceding photoperiod. Nyctoperiods ofup to 9 hr following a 12 hr photoperiod hardly affected flowering,but nyctoperiods given after a 24 hr photoperiod suppressedflowering in proportion to the length of the period. The Pfr-levelin the nyctoperiod, therefore, seems to be important for flowering,and phytochrome change, as a function of the length of photoperiod,may serve as a photoperiodic timer. Although floral responseto interruption with R or FR changed with the application period,the difference in response between R-treated and FR-treatedplants was relatively constant during a 15 hr nyctoperiod combinedwith a photoperiod of any length other than 1 hr. Apparently,the floral response to the R or FR pulse was regulated by ashift in the Pfr-level caused by the light pulse. (Received April 2, 1979; )     

A "Point of No Return" in the Cell Cycle of Chlorella

In a Chlorella culture growing synchronously at pH 6.3 undera 12 hr light/12 hr dark regime, DNA replication occurs betweenthe 8th and the 12th hour of the cycle, the main period of proteinand chlorophyll synthesis occurring between the 4th and 12thhour of the cycle. When the culture is transferred to alkalinepH at any time up to the 8 hr of the cycle, autospore releaseis prevented, and the pattern of synthesis of DNA, protein andchlorophyll is altered. However, when the culture is transferredto alkaline conditions after the 8th hour of the cycle, thepattern follows that of a culture growing at pH 6.3 with respectto cell number and volume, as well as protein, chlorophyll andDNA contents. Thus, a transition point seems to occur afterthe 8 hr of the cycle. The existence of such a point was alsodemonstrated by reciprocal experiments in which Chlorella wascultured at an alkaline pH and transferred to pH 6.3 at varioustimes in the cell cycle. ¹ Present address: Applied Research Institute, Ben-Gurion Universityof the Negev, P.O. Box 1025, Beer-Sheva 84110, Israel. (Received October 2, 1981; Accepted January 20, 1982)     

Perithecial formation in Gelasinospora reticulispora III. Inhibitory effects of near-UV and blue light during the inductive dark period

Growing hyphae of Gelasinospora reticulispora required a continuousdark period prior to photoinduction of perithecia. The inductivedark period was interrupted by brief exposure of the hyphaeto white light so that the formation of perithecia no longertook place. Photosensitivity of the hyphae in terms of the light-breakeffect gradually changed during the inductive dark period. Sensitivityreached its maximum at the 18th hr of the dark period when anirradiation of 1?10⁵ ergs cm^–2 of near-UV light or 4?10⁴ergs cm^–2 of blue-light was sufficient for the light-break.Red and far-red light had no effect at all. The light-breakeffect was limited to the irradiated portion of the hyphae anddid not affect any unirradiated portions. Inhibitory effecton perithecial formation of continuous white light could betotally replaced for several days with intermittent irradiationof near-UV or blue light if given for 5 min every 4 hr. (Received December 18, 1973; )     

Endogenous light-on rhythm in respiration of a long-day duckweed, Lemna gibba G3 II. On basic and rhythmic components of the rhythm

Effects of respiratory substrates (glucose, malate, citrateand pyruvate) and inhibitors (fluoride, iodoacetate, azide andDNP) on the O₂-uptake rhythm in a long-day duckweed,Lemna gibbaG3 in continuous light period were examined. Rates of O₂-uptake at the starting point (6 hr after the beginningof a continuous light period) and at the time of the first peakof the rhythm (18 hr after the beginning of a continuous lightperiod) were equally increased by exogenous substrates. Sensitivityof respiration to fluoride or iodoacetate was almost the sameat the 6th and 18th hr. The O₂-uptake (at the 6th, 18th, 30thand 42nd hr) was increased by DNP by the same amount. Azideat lower concentrations than 5X10^–4 M did not affect O₂-uptakeat the 6th hr, but inhibited uptake at the 18th hr. In the presenceof 5 X 10^–4 M of azide the rates of O₂-uptake at the 18th,30th or 42nd hr were down to the rate at the 6th hr, which wasinsensitive to azide. These results suggest that the O₂-uptakerhythm consists of two components, i.e. the basic respirationwhich is promoted by exogenous substrate, sensitive to DNP andinsensitive to azide; and rhythmic respiration, which is sensitiveto azide, but is not influenced by exogenous substrate and DNP. (Received February 19, 1971; )