Rhythms in respiratory metabolism of Lemna gibba G3 under continuous illumination

Changes in chemical constituents and respiratory metabolismof a long-day duck-weed, Lemna gibba G₃ exposed to continuousillumination after short-day cultivation were investigated.The dry weight to fresh weight ratio was constant during thefirst 72 hr of continuous illumination. pH of the crude extractwas constant at 6.6, but pH of the culture medium was raisedwith the Lemna growth. Titratable acidity decreased after about44 hr, whereas malic acid content increased in 18 hr. Therewere no significant changes in total reducing sugar and pentose.Total protein content and lipid showed rhythmical changes withcycles of 48 hr. O₂-Uptake gave a damped oscillation with cycles of 24 hr. Itwas low in the first half day and high in the second half. ¹⁴CO₂-Outputfrom glucose-l-¹⁴C showed a similar damped oscillation. ¹⁴CO₂-Outputfrom glucose-2-14^C or glucose-6-₁₄C was almost constant. TheC₆/C₂ ratio, then, showed damped oscillation in the reverseway to O₂- uptake between 0.3–0.5, and the C₈/C₂ ratiowas constant at 0.9. Accordingly, the diurnal rhythm of O₂-uptakewas thought to be brought about by variation in activity ofthe pentose-phosphate pathway. Reproduction of glucose-6-phosphateby the pentose-phosphate pathway was presumably limited in amount. Glyceraldehyde-3-phosphate dehydrogenase activity varied diurnally.The activities of NADP-linked and NAD-linked enzymes increasedand decreased, respectively, in the first half day. Variationsin these enzymatic activities are discussed in correlation withrhythmical changes in O₂-uptake and in the C₆/C₁ ratio. Acidphosphatase activity also followed a diurnal variation. No activitiesof alcohol and formic dehydrogenases were found. The activitiesof NADP glucose-6-phosphate dehydrogenase, 6-phosphogluconatedehydrogenase, pyruvic kinase and NADP isocitric dehydrogenasewere high, but showed no rhythmical variation. ¹Presented at the Annual Meeting of the Botanical Society ofJapan, 1966 (Proceedings, p. 46). Adapted from a thesis submittedby the first author (H. M.) in 1967 to the Biological Institute,Nagoya University in partial fulfillment of the requirementsfor the degree of M. S. (Received May 8, 1969; )     

The occurrence of acetylcholine in Lemna gibba G3

The occurrence of acetylcholine in a long-day duckweed, Lemnagibba G3 has been demonstrated. After a preliminary purificationof the formic acid-acetone extract by Sephadex G-15 column chromatography,acetylcholine was identified by paper chromatography, pharmacologicalactivity on frog muscle, and sensitivity to acetylcholinesterase. Acetylcholine contents relative to that at the start of theexperimental culture were 0.99?0.06, 1.61?0.27, and 1.17?0.16after 2 cycles of the [9(15)], [16(8)], and [24(0)] schedules,respectively. (Received November 14, 1977; )     

Induction of flowering in Lemna gibba G 3 by Fe-EDDHA

Fe-EDDHA (iron salt of ethylenediamine-di-o-hydroxyphenylaceticacid) induced profuse flowering in Lemna gibba G 3 culturedin HUTNER'S medium. The maximum number of flowering plants wasobserved in a medium supplemented with 5 ppm of this chelate. (Received April 20, 1970; )     

Gibberellin-EDDHA interaction in flowering and gibbosity of Lemna gibba G3

The effect of gibberellic acid (GA₃), in the presence of EDDHA,on the flowering and gibbosity of Lemna gibba G3 was studied.At 10 ppm and at higher concentrations of GA₃ the EDDHA-effect,i.e. profuse flowering and conspicuously gibbous fronds, wascompletely nullified. (Received July 15, 1974; )     

Periodical growth response of Lemna gibba G3 to light-break

Brief exposure to light promotes frond multiplication in Lemnagibba G3 in darkness. Extent of promotion changes periodicallywith the time of the light-break. Response curves are interpretedin terms of a superposition of two modes of growth responseto light-break, which are, respectively, under the control ofdifferent physiological timing devices; circadian oscillationand the hourglass-type clock. Circadian oscillation, which consistsof a half-cycle of increasing photophily followed by anotherhalf-cycle of declining photophily, starts at a light-on signaland continues for a few days with rapid damping. The 24-hr periodof oscillation is the same at temperatures ranging from 16 to26°C. The hourglass is released by a light-off signal to‘accumulate sand’ or to increase photophily in asigmoidal way with time and is temperature-sensitive; the tempoof‘sand accumulation’ being quicker at 21°Cthan at 16 or 26°C. Oscillation is hastened to fade-outat 21°C, most likely due to the accelerated pace of thehourglass. Red and far-red reversibility is disclosed in bothmodes of growth response. (Received December 31, 1969; )     

Flowering Responses of the Long-day Plant Lemna gibba G3

Lemna gibba L., strain G3, exhibits a qualitative long-day flowering response with a critical daylength on a 24-hour cycle of about 10 hours. Evidence is presented that the onset of daughter frond formation in a given frond inhibits the activity of the flowering meristem. Consequently, flower induction can only occur in fronds smaller than about 0.05 to 0.07 mm long. Although a minimum of 1 long day seems to be sufficient to induce the formation of flower primordia, at least 6 long days are required to obtain mature flowers since long days are also required for the early stages of flower development. The critical night length on 24, 48 and 72-hour cycles is respectively 14, 16, and 18 to 22 hours. The close similarity between the critical night length for the different cycle lengths is explained in terms of an inhibitory effect of darkness both on flower initiation and flower development. A 10-hour dark period is more inhibitory to flowering on a 36-hour cycle than on 24, 48, 60 or 72-hour cycles. It is suggested that darkness inhibits flowering through the formation of a light-labile flower inhibitor which acts to inhibit the functioning of the flowering stimulus.     

The response of Lemna gibba G3 to a single long day in the presence of EDTA

Lemna gibba G3 in M-EDTA or M-EDTA-sucrose medium initiatesflowers in response to a single long day, but requires a minimumof nearly or exactly 2 long days in an M or M-sucrose mediumfor flower induction. (Received December 18, 1971; )     

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; )     

Induction of flowering in Lemna gibba G3 under short-day conditions

In the presence of the chelating agent EDDHA, long-day duckweedLemna gibba G3 was induced to flower under a short-day scheduleof 9 hr of light and 15 hr of darkness in a 24-hr cycle. Weconcluded that EDDHA creates effects very similar to those ofsalicylic acid. When EDDHA or salicylic acid was added to thenutrient medium in combination with BA, flowering was inducedeven under conditions of 8 hr of light and 16 hr of darkness.Under a photoperiod of 9 hr, BA markedly enhanced the effectof EDDHA as well as salicylic acid. On the other hand, BA alonewas ineffective as far as flowering was concerned. By quantitativeinteractions, BA seems to complement the modifying effect ofEDDHA or salicylic acid on flowering in this duckweed strain. (Received June 25, 1976; )     

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

Short-day flowering of Lemna gibba G3 induced by salicylic acid

Salicylic acid, probably as a chelating agent of the EDTA-salicylaldoximetype, can eliminate the light requirement during the inductivephase of Lemna gibba G3, and thus is able to induce short-dayflowering of this long-day plant. (Received September 4, 1975; )     

Lack of Correlation between Senescence and Stomatal Aperture in Lemna gibba G3

Stomata on the frond of Lemna gibba G3 were found to be non-functional;they remain open regardless of treatments that lead to eitherpromotion or inhibition of frond senescence. These findingsare not in accordance with the notion that leaf senescence isprimarily mediated through the stomatal behavior. ¹Present address: Department of Botany, North Carolina StateUniversity, Raleigh, North Carolina 27695, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

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; )     

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.     

Removal by chemicals of photoperiodic light requirements of Lemna gibba G3

Both the initial and the terminal 1 hr portions of the subjectiveday fraction, namely the L1- arid L2-phases, of a 24 hr daymust be illuminated in order for the day to be perceived asa long day in the min-LD determination by the long-day plant,Lemna gibba G3 (9). The light requirement of the L1-phase wassatisfied by a 10 min red light pulse given at the beginningof the phase. The red light effect was erased by a subsequent10 min far-red light, indicating phytochrome-mediated processesoccurring in the L1-phase. The light requirement of the L2-phasewas satisfied by blue or far-red light given during the terminal10 min period of the phase; there was no indication of phytochromeinvolvement. The light action on the L1-phase was replaced by10^–5 M of cyclic AMP or 10^–7 M of DL-isoproterenol.The isoproterenol action was antagonized by 10^–7 M ofDL-propranolol. Cyclic AMP (10^–5 M) combined with salicylicacid (10^–6 M), which can remove the light requirementof the L2-phase (10), rendered a completely dark day physiologicallyequivalent to a long day. Acetylcholine (10^–5 M) exertednyctomimetic action on the L1-phase of the second light day.The action of acetylcholine was antagonized by cyclic AMP (10^–5M). The L2-phase required no light in the presence of 10^–7M of DL-propranolol, and this propranolol action was not affectedby isoproterenol. These findings suggest changes in membranepermeability caused by the light given during the L1- and L2-phases. (Received July 7, 1976; )     

Characterization of Senescence in Lemna gibba G3: A Determinate Growth System

Frond senescence in Lemna gibba G3 was characterized, and itscontrol by light, ABA and kinetin investigated. The plant exhibitsa determinate growth pattern with a frond producing a set numberof daughter fronds before undergoing senescence and death regardlessof whether or not it flowers. When a frond was cut in half,the distal half (half frond) which lacks any meristem underwentrapid senescence as compared with intact fronds. In both intactand half fronds, the onset of senescence was accelerated byABA and retarded by kinetin. Continuous white light acceleratedsenescence in both intact and half fronds over the dark controls.Under different photoperiodic light regime, the pace of daughterfrond production is accelerated in proportion to the lengthof light period. In half fronds, however, very short photoperiodiclight treatments (e.g. 1L: 23D or 3L: 21D) rather delayed senescenceover the dark controls. Two separate light control systems operatingin opposite directions in Lemana senescence appear to exist. ¹Present address: Department of Biology, Yonsei University,Seoul 120-749, Korea ²Present address: U.S. Department of Agriculture, Aero SpaceBuilding, Rm. 323, 901 D Street, S.W., Washington, D.C. 20251-2200, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

Replacement by ionophores of the photoperiodic light requirement in Lemna gibba G3

Light requirement during the phytochrome-mediated L1-phase ofLemna gibba G3 can be replaced by 10 µg/liter of valinomycinor gramicidin, supporting the idea (5) that a promoted K⁺-iontransport across membranes would be involved in the photoperiodiclight action on the L1-phase. (Received June 6, 1977; )     

Uptake and Metabolism of [C]Salicylic Acid in Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH₄⁺-free 0.5 H medium) there is virtually no flowering, but addition of 10 micromolar salicylic acid (SA) to the medium results in substantial flowering. Using this system, the uptake and metabolism of [¹⁴C]SA in L. gibba G3 has been examined. SA uptake is rapid and linear for at least the first 24 hours. After 30 minutes, nearly 90% of the radioactivity in the plants is present as free SA. Part of this is rapidly converted to one or more bound forms of SA that appear either in the acidic butanol fraction or in the aqueous residue, and after 12 hours an equilibrium is reached between the free and bound forms of SA. When plants receive SA for 6 days and then are switched to control medium, both the free and bound SA remain nearly constant for at least 5 days. However, there is virtually no transfer of SA from mother fronds to daughter fronds, indicating that the SA is apparently sequestered within the cell. Cell fractionation studies show that nearly 95% of the SA remains in the supernatant even after a 2-hour centrifugation at 300,000 g. Thus, it is unlikely that SA is confined within a specific organelle, but rather is probably secreted into the vacuole.     

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; )     

Specific interactions in the physiology of flowering and gibbosity of Lemna gibba G3

Fronds of Lemna gibba G3 became conspicuously gibbous when ethrel,an ethylenereleasing compound, was added to the nutrient medium.Maximal gibbosity was obtained at ethrel concentrations of 1µg/ml and higher. Unlike the chelating agent, EDDHA, whichcauses profuse flowering and markedly gibbous fronds under long-dayconditions, ethrel did not affect flowering. In the presenceof an optimal concentration of EDDHA (10 µ/ml), ethreleven significantly inhibited flowering and caused developmentof excessively gibbous fronds. Autoclaved gibberellic acid specifically negated the ethreleffect as it does that of EDDHA. Three decomposition productsof GA₃, allogibberic acid, epiallogibberic acid and gibbericacid, also nullified flowering and gibbosity in the presenceof EDDHA. A fourth decomposition product of GA₃, epigibbericacid, inhibited gibbosity but hardly affected flowering. Salicylic acid was confirmed to affect flowering and gibbosityin L. gibba G3. However, contrary to an earlier report, it didnot induce flowering under short-day conditions. (Received January 10, 1976; )