Endogenous light-on rhythm in respiration of a long-day duckweed, Lemna gibba G3 III. Relationship to frond production

Relationship between the C>2-uptake rhythm and frond productionin a long-day duckweed, Lemna gibba G3, was investigated. The rate of frond production and the amplitude of the rhythmwere dependent on light intensity. Photosynthetic inhibitors,CMU and DCMU, at concentrations effective in stopping frondproduction, abolished the O₂-uptake rhythm after a lag of 1day. In the presence of inhibitors of protein and RNA syntheses,ETH, CH and TU, at concentrations which brought about completeinhibition of frond production, the O₂-uptake rhythm disappeared.FUdR, an inhibitor of DNA synthesis, did not eliminate the rhythmalthough it suppressed frond production. This indicates a ratherindirect correlation between the rhythm and the rate of frondproduction which, in turn, is probably related to photosynthesis.The rhythm may be more directly correlated with the cell expansion. (Received July 27, 1971; )     

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; )     

Uptake of uridine by a long-day duckweed, Lemna gibba G3

Uptake of uridine by a long-day duckweed, Lemna gibba G₃ wasexamined. Km and Vmax for uptake were in the range of 1 to 2x10^–5 M and of 5 to 10 x10^–8 moles/g fresh weight/2hr, respectively. Uptake rate depended on temperature, and theoptimum pH was 5.0. Uridine uptake was competitively inhibitedby some compounds structurally analogous to uridine. However,the activity of uridine kinase was not affected by these compounds,except for cytidine. Uridine uptake was inhibited by metabolicinhibitors, in which uridine taken up was left unconverted toother forms, especially in the presence of DNP. These resultssuggest that uridine was taken up into the duckweed celb bya specific transport system and immediately phosphorylated byuridine kinase. Phosphorylation of uridine was not associatedwith the uridine transport reaction. (Received November 15, 1976; )     

On the rhythm of sensitivity to light interruption in a long-day duckweed, Lemna gibba G 3

1. The rhythm of sensitivity to light interruption in a long-dayduckweed, Lemna gibba G 3, was examined. The rhythm was circadianand was suggested to be under the control of a physiologicalclock. Light given in the trough between the first and secondpeaks reset the rhythm. Five to 7 cycles of non-circadian photoperiodicregimes given entrained the rhythm to external periodicity,and this entrained rhythm persisted even after the plants weretransferred to continuous darkness. 2. It was suggested that the induction period is not determinedby the physiological clock disclosed here, but by a periodicalternation of dark-sensitivity, or by a periodic change inactivity of SS (system sensitive to IPEF, induction period extendingfactor,) as postulated previously. (Received November 28, 1967; )     

Photoperiodic requirements for flowering of the long-day duckweed, Lemna gibba G3

The min-LD estimation by the log. flower number vs. the cultureperiod curve provides a unique method of judging whether a givenphotoperiodic schedule is a long day or not for Lemna gibbaG3. Duckweeds in M-sucrose medium are exposed to the test scheduleat 26°C on either the first or second day of a continuouslight culture. If the min-LD (48 hr for control cultures) isnot changed, the inserted schedule is considered to have functionedas a long day. If, however, the min-LD is extended by 24 hr,the inserted schedule is judged to have functioned as a shortday. Examinations using this method of orderly designed light-darkschedules disclosed two critical phases in the light requirement;the initial and terminal 1 hr portions (designated the L1- andL2-phases) of the subjective day. Thus, a given day became along or short day when both the L1- and L2-phases were illuminatedor when either or both of the two phases were darkened. Thecritical daylength (11.5 hr) was just long enough to cover boththe L1- and L2-phases and the inductive phase (L2-phase) waslocated at the end of the subjective day. (Received June 9, 1975; )     

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; )     

Effects of some metabolic inhibitors on flowering of Lemna gibba G3, a long-day duckweed

Flowering of Lemna gibba G₃, a long-day duckweed, was inhibitedby adding CuSO₄, AgNO₃, HgCl₂, Na₂WO₄ or iodoacetamide to themedium at the concentrations inducing long-day flowering inLemna paucicostata 6746, a short-day duckweed. This suggeststhat these metabolic inhibitors affected the photoperiodic sensitivityrather than directly affecting flower initiation. Ferricyanidepromoted flowering in both of these short-day and long-day duckweeds. (Received July 7, 1977; )     

Spectral dependence of the critical photoperiod in the long-day duckweed, Lemna gibba G3

Spectral dependence of the light sensitive L1- and L2-phasesof the critical photoperiod of Lemna gibba G3 was examined bythe min-LD method for wavelengths ranging from ca. 420 to 800nm. The L1-phase had a sharp peak of sensitivity at 650–680nm and a gentle slope of sensitivity ascending from 500 nm towardshorter wavelengths. The L2-phase showed remarkable sensitivityaround 750 nm and in the blue-violet regions (500 nm). Red/far-redphotoreversibility was confirmed for the LI-phase, but not forthe L2-phase. The properties of the light receptors involvedare discussed briefly. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan. (Received May 14, 1979; )     

Energy supply for potassium uptake rhythm in a duckweed, Lemna gibba G3

The energy relationships of the potassium uptake rhythm in aflow-medium culture of a duckweed, Lemna gibba G3, were investigated. Respiratory inhibitors or uncouplers (NaCN, 2,4-dinitrophenoland carbonyl-cyanide m-chlorophenylhydrazon) reduced the mesoror the average rate of potassium uptake, without remarkablydecreasing the amplitude of the rhythm. NaN₃, however, preferablysurpressed the amplitude at a concentration as low as 10^–6M. Both dichloro-phenyldimethylurea and the removal of CO₂ graduallydecreased the average rate of the uptake, although the rhythmicityitself was not eliminated completely in the absence of CO₂.In N₂, the average rate of uptake was reduced to zero, but rhythmwith a small amplitude survived. These observations suggested that respired photosynthates provideenergy for sustaining the average of potassium uptake and thatsome light dependent processes control the amplitude of therhythm. Exogenous sugar could make the potassium uptake rhythmappear for a relatively short time in the dark. ¹This study was carried out in the National Institute for BasicBiology and the Biological Institute, Faculty of Science, NagoyaUniversity (Received October 24, 1979; )     

Further studies on the diurnal rhythm of uridine incorporation into RNA in the long-day duckweed, Lemna gibba G3

Using the long-day duckweed Lemna gibba G3, the changes in theactivities of RNA synthesis in isolated nuclei and chloroplastsand of the reaction prerequisite for the incorporation of exogenousuridine into RNA were examined. When the duckweed was exposedto either a light-dark cycle or to continuous light, the activityof RNA synthesis in the nuclear and chloroplast fractions changeddiurnally and reached its highest levels during the night phase.The changes coincided with uridine incorporation into RNA invivo. However, the amount of radioactive uridine taken up intothe acid-soluble fraction remained unchanged during the wholeday. The proportion of radioactivity incorporated into phosphorylateduridine compounds as well as UTP+UDP to the radioactivity inthis fraction remained constant. Thus, the diurnal rhythm ofuridine incorporation into RNA was related to the diurnal rhythmof RNA synthesis in isolated nuclei and chloroplasts. The loweractivity of uridine incorporation into RNA under continuousdarkness may be determined by the activity of RNA synthesisin nuclei and chloroplasts as well as the uptake rate of uridineinto the duckweed cells, not by the activity of its phosphorylation. (Received August 30, 1977; )     

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; )     

Further studies on potassium uptake rhythm in the long-day duckweed Lemna gibba G3 with special reference to vegetative growth

Some properties of the circadian rhythm in potassium uptakeof flow medium culture of the long-day duckweed Lemna gibbaG3 were examined.

1. In total darkness, the rhythm faded out in ca. 48 hr; it restartedon transfer to continuous light. Under low-intensity light (below700 lux), the rhythm was damped rapidly
2. The rhythm appearedregardless of the potassium concentrationin the culture medium(from 10/m to 2 HIM). The amplitude, butnot the period, ofthe rhythm was influenced by the ambientpotassium concentration.
3. Alteration in the light intensity or medium composition causeda change in the growth rate without modifying the period ofthe rhythm.
4. These results indicate that potassium uptake rhythmin thisduckweed is typical light-on rhythm, which has no directrelationwith the rate of vegetative growth and requires lightenergyfor its duration.

¹Present address: Aichi-Gakuin University, Chikusa-ku, Nagoya464, Japan. ²Present address: National Institute for Basic Biology, Okazaki,Aichi 444, Japan. (Received February 1, 1979; )     

Effect of light on the metabolism of thymidine in the long-day duckweed, Lemna gibba G3

Metabolism patterns of exogenous thymidine as disclosed by ECTEOLAcellulose column chromatography, were examined with a long-dayduckweed, Lemna gibba G3, under dark and light conditions. Whenthymidine-6-³H was applied, the pattern of thymidine metabolismin the light was not very different from that in the dark. However,when thymidine (methyl-³H) was used, incorporation of radioactivityinto two major ( and ß) and one minor components ofboth B and D fractions separated by column chromatography, wasstrikingly stimulated by the light, through photosynthetic activity.Component a of fraction B was tentatively concluded to be ß-ureidoisobutyricacid by paper chromatography. As the radioactivity from thymidine-6-³Hwas hardly recovered in the a component of fraction D, the partof the thymidine molecule incorporated into this component wasnot the pyrimidine ring, but a methyl residue. (Received March 29, 1973; )     

Participation of a membrane system in the potassium uptake rhythm in a duckweed, Lemna gibba G3

The period of the free-running potassium uptake rhythm in along-day duckweed, Lemna gibba G3, was lengthened by Li⁺ andshortened by ethanol. This suggests that a membrane system participatesin the circadian feed-back system. High temperature treatment(39°C) increased the motion of the rhythm when applied duringperiods of high potassium uptake (CT 18–06), and decreasedit when applied during low potassium uptake (CT 06–18),which indicates a circadian change in the properties of themembrane. However, modification of the potassium uptake level caused bytemperature change, application of valinomycin, or a changein the ambient potassium concentration caused no modificationin the phase of the rhythm. Thus, the oscillator involved probablyis not located in the cell membrane or the ectoplasm. ¹National Institute for Basic Biology, Myodaiji, Okazaki 444,Japan. ²Aichi-Gakuin University, Chikusa, Nagoya 464, Japan. (Received October 24, 1979; )     

Rhythm in potassium uptake by a duckweed, Lemna gibba G3

The potassium uptake activity of the "flow-medium culture" ofa long-day duckweed, Lemna gibba G3, followed a circadian rhythmwhich persisted for more than 5 days under continuous light.The period of the rhythm was about 25 hr under 3000 lux at 26?Cand was slightly over-compensated against temperature, Q₁₀ beinga little less than 1.0. The amplitude of the rhythm was dependenton light intensity, and there was no potassium uptake in thedark. Magnesium uptake was affected by the potassium movementand showed circadian rhythmicity with a small amplitude underconditions where the potassium uptake was already saturated.Calcium uptake did not show any obvious rhythm. In Contrastto L. gibba, a short-day duckweed L. perpusilla 6746 displayedcircadian rhythm of potassium uptake only in the dark and notin the light. This rhythm did not persist beyond the secondcycle. (Received June 13, 1978; )     

Reversal of the dark inhibition of flowering in a long-day duckweed, Lemna gibba G3, by thymidine and related nucleosides

Effects of several nucleosides on flowering in Lemna gibba G3inhibited by darkness inserted just before an inductive lightperiod were investigated. Thymidine and deoxyuridine could reversethe inhibition, but deoxycytidine, cytidine, uridine, thymineand deoxyribose could not. Since interruption of die darknesswith a brief light period was effective similarly to additionof thymidine and deoxyuridine, the light-stimulated step inthymidine biosynthesis is probably the reaction of deoxyuridinesynthesis. Furthermore, maintenance of thymidine content isprobably required for the progress of floral induction. (Received March 29, 1973; )     

Phase progress under low temperature treatment of the potassium uptake rhythm in a duckweed, Lemna gibba G3

Potassium uptake rhythm in the long-day duckweed Lemna gibbaG3 grown at 26?C disappeared at temperatures below 12?C. However,when the plants were returned to 26?C, the rhythm immediatelyrestarted from circadian time 12 with its normal wave form.Temperature steps from 20 to 30?C or from 30 to 20?C did notmodify the phase of the rhythm, although a step from 15 to 30?Cor from 30 to 15?C evoked a distortion in the wave form withoutintroducing any reproducible phase shift. Various periods of 9 or 4?C given during the subjective dayphase reduced the pace of rhythm progress by 40 or 60%, whilethose given during the subjective night phase did not. Theseresults suggest that the subjective day and night phases arethe energy charge and discharge phases for the underlying oscillator,respectively. Energy fluxes for the oscillators are brieflydiscussed. ¹Present address: National Institute for Basic Biology, Okazaki444, Japan. ²Present address: Aichi Gakuin University, Chikusa, Nagoya 464,Japan. (Received August 25, 1979; )     

Diurnal change in leakage of electrolytes from a long-day duckweed, Lemna gibba G3, under osmotic stress induced by water treatment

The leakage of electrolytes from Lemna gibba G3 placed on waterwas investigated in terms of the change in the electroconductivityof the ambient water. Amounts of the leaked electrolytes reacheda maximum 5–6 hr after transfer to water, followed bygradual decrease. The maximum amount of the leakage changeddiurnally, with the time of the water treatment, with a peakin the early subjective day and a trough in the early subjectivenight. The rhythm was reset by a light-on signal, persistedfor at least 3 days under continuous light, and its period wastemperature-compensated. The rhythm was maintained even in thepresence of 2,4-dinitrophenol or 3-(3,4-dichlorophenyl) 1,1-dimethylurea and under weak light, suggesting that a changein passive efflux of electrolytes was involved, and this wasconfirmed by kinetic studies which revealed that the leakageoccurred by simple diffusion. Strong illumination extinguishedthe rhythm, probably by promoting the active reabsorption ofthe leaked ions. Potassium ions were the major cations found,to the leakage of which was attributed half of the change inthe electroconductivity of the ambient water. (Received January 18, 1978; )     

Nodular somatic embryogenesis and frond regeneration in duckweed,Lemna gibba G3

Duckweed(Lemna gibba) is a useful model system for elucidating plant development, but the techniques needed for regenerating fronds from calli are not yet well established. This study examined the effects of auxin, sucrose, and gelling agents on callus and frond formation inL. gibba G3. After three weeks of culturing on a solid medium, two types of calli were observed: watery, pale-green, and undifferentiated; or white, compact calli that were organized into nodules and which resembled somatic embryogenie calli. Homogeneous callus lines were produced through selective subculture. To induce nodular calli, auxin (2,4-D) was absolutely required, with an effective concentration of 5 to 20 μM; induction was found to be possible with up to a maximum concentration of 4.4%. The calli were then maintained on a medium with a reduced 2,4-D concentration (1 μM), and were transferred every three weeks. Optimal callus induction and growth were obtained by using 3% sucrose with a combination of 0.15% Gelrite and 0.4% agar. Fronds, however, could be regenerated only on distilled water solidified with a combination of 0.4% agar and 0.15% Gelrite. On this medium, 87% of the callus expiants regenerated into fronds after four weeks of culture. These new fronds were morphologically normal but small, approximately 15 to 20% of the size of stock fronds. Continued culture of these fronds in an SH medium produced normal duckweeds, and histological examination of the cultures revealed several distinct types of callus nodules. Nonetheless, because zygotic embryogenesis inL. gibba does not produce distinct bipolar structures, the developmental pathway of frond regeneration from these nodular cultures remains unknown.     

Diurnal rhythm of nuclear RNA polymerase I activity in a duckweed, Lemna gibba G3, under continuous light conditions

The diurnal change of nuclear RNA polymerases I and II was examinedin a longday duckweed, Lemna gibba G3, under continuous lightconditions. RNA synthesis in crude nuclei was dramatically stimulatedby addition of the exogenous RNA polymerase of Escherichia coli,but not by the addition of calf thymus DNA. Treatment of crudenuclei with the supernatant fractions after precipitation ofthe nuclei decreased the RNA synthetic activity of the nucleiirrespective of the preparation time of both fractions. RNApolymerase I activity in crude nuclei, which was determinedin the presence of -amanitin at a low concentration of KCl,exhibited a diurnal rhythm but RNA polymerase II activity, whichwas presumed to be a portion of RNA synthesis inhibited by -amanitinin the presence of a high concentration of KCl, remained constantthroughout the day. Identical results were obtained when bothenzymes were solubilized widi ammonium sulfate and chromatographedon DEAE-Sephadex column. Both activities in the supernatantfraction obtained after precipitation of the nuclei did notchange diurnally. It was concluded, therefore, that the diurnalrhythm of RNA synthetic activity in the crude nuclei is dueto the RNA polymerase I activity and not the RNA polymeraseII activity. (Received August 29, 1978; )