Effect of L-Pipecolic Acid on Flowering in Lemna paucicostata and Lemna gibba

L-Pipecolic acid was found to be effective in inducing floweringof Lemna paucicostata 151, 381, 441 and 6746, and of Lemna gibbaG3. When the plants were grown on half-strength Hutner's medium,L-pipecolic acid caused profuse flowering of L. paucicostata151 maintained under 9 and 10 h of light daily. In L. paucicostata441 and 6746, L-pipecolic acid had a strong flower-promotingeffect under a near critical photoperiod. In L. paucicostata381, by contrast, L-pipecolic acid had only a very small effecton flowering. In L. gibba G3 substantial promotion of floweringwas observed under continuous light. When one-twentieth-strengthHutner's medium was used as the basic medium, L-pipecolic acidstimulated flowering in all strains of Lemna examined, evenunder continuous light. When L. paucicostata 151 was grown on one-tenth-strength M mediumor one-twentieth-strength Hutner's medium, the flower-inducingactivity of L-pipecolic acid was greatly enhanced by cytokininunder continuous light. However, when this strain was grownwith 9 h of illumination daily, this synergistic effect of cytokininwas only slight. A short-term (even 1-h) treatment with L-pipecolicacid resulted in flowering, suggesting that L-pipecolic acidis involved in the induction of flowering, rather than its evocation.D-Pipecolic acid also had flower-inducing activity, but itsactivity was 50 times lower than that of the L-isomer. (Received January 23, 1992; Accepted March 9, 1992)     

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.     

Influence of Giving Salicylic Acid for Different Time Periods on Flowering and Growth in the Long-Day Plant 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) growth is excellent, but flowering is severely inhibited and often is zero. Addition of 10 micromolar salicylic acid (SA) to NH₄⁺-free 0.5 H medium quickly reverses this inhibition and leads to optimal flowering. The SA treatment also leads to a considerable reduction in the growth rate and increase in frond gibbosity. Removal of SA from the medium quickly leads to an increase in the growth rate and a large decrease in flowering. Thus, for maximal effectiveness SA must be present in the medium for the entire experiment, and the effect of SA is clearly not inductive.     

Growth Cycles in Lemna gibba Cultures and Their Effects on Growth Rate and Ultrastructure

A long-established axenic culture of Lemna gibba G3 was maintained in exponential growth phase under controlled conditions. Weekly analyses for 2 years showed that the individual plants of the Lemna gibba clone fluctuated between two forms. One extreme consisted of plants light in weight, small in size, and with a high relative growth rate (RGR), the other of heavy, large, and more slowly growing plants. At intervals plants having intermediate characteristics dominated in the stock culture. Indication of an annual growth-cycle was also found. The magnitude of growth response (weight, RGR, area, and dry matter content) after treatment with abscisic acid (ABA), 6-benzylaminopurine (BAP), and a combination of the two was different for low-weight and heavy plants. The heavy plants were more sensitive to ABA and BAP treatment than the low-weight ones. The accumulation of starch was least in small untreated plants and greatest in ABA treated plants. Large electron transparent globules were found in the chloroplasts of the ABA treated plants and in heavy plants regardless of how they had been treated. The different physiological and ultrastructural characteristics of the two forms of Lemna plants probably reflect an ageing-rejuvenation cycle. Emphasis is placed on the importance of this cycle when Lemna is used as a model plant in physiological experiments.     

Genetic transformation of duckweed Lemna gibba and Lemna minor

Summary We developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.     

Growth potential of Lemna gibba: Effect of CO2 enrichment at high photon flux rate

Lemna gibba L. was cultivated in continuous light (800–1200 μmol quanta m^?2s^?1, 320–400 W m^?2) and normal or CO₂-enriched air (1500 μl CO₂ l^?1), with a continuous nutrient supply. Increased CO₂ concentration increased the unit leaf rate (ULR) or net assimilation rate and decreased the leaf area ratio (LAR) (photosynthetic area per unit dry weight), but the relative growth rate was unchanged (0.43 g g^?1 day^?1). The changes in ULR and LAR indicate that organic matter production can be increased with CO₂ enrichment at high photon flux rate (PFR).     

The Role of Benzoic Acid and Plant Hormones in Flowering of Lemna gibba G3

The level of benzoic acid was measured in Lemna gibba G3 grownon M and E media under inductive and non-inductive daylengths.Benzoic acid was slightly higher in plants grown on M mediumbut there was no difference in the benzoic acid levels in floweringand vegetative plants. When L. gibba G3 was grown under continuouslight on 1/10 M medium or 1/2 H medium there was virtually noflowering, but addition of benzoic acid to either medium ledto a substantial flowering response. In both cases this floweringresponse was inhibited by the plant hormones IAA, GA₃, ABA andzeatin, with IAA and GA₃ being the least inhibitory and ABAbeing the most inhibitory. This same pattern of inhibition wasseen when L. gibba G3 was grown on M medium under continuouslight, conditions that lead to photoinduction of flowering.These results leave open the possibility that endogenous benzoicacid may interact with other factors to influence the floweringresponse in L. gibba G3. (Received November 13, 1984; Accepted February 27, 1985)     

Effect of circulation on wastewater treatment by Lemna gibba and Lemna minor (floating aquatic macrophytes)

In this study, laboratory tests were performed in order to examine growth characteristics of floating aquatic macrophytes (Lemna gibba and Lemna minor) in the presence of wastewater with circulation. The results showed that circulation of the waste water enhanced the kinetics of the process, as compared to the control systems. However, prolonged application of high circulation level had a different effect. In the presence of circulation with aquatic plants, there was additional 85.3-88.2% for BODs and 59.6-66.8% for COD decreases in the water quality indicators. In this study, the effectiveness of L. gibba and L. minor with circulation addition for the removal of four heavy metals (Pb, Ni, Mn, and Cu) from waste water was also investigated. Results from analysis confirmed the accumulation of different metals within the plant and a corresponding decrease of metals in the waste water. At the end of the study of circulation, L. gibba provided the metal removal for Cu, Pb, Ni, and Mn in the waste water as the ratio of 57%, 60%, 60%, and 62%, respectively. In this context, the best results were obtained when the action of L. gibba and L. minor plants, was combined with that of circulation. It is shown that in the presence of L. gibba and L. minor plants that are supplemented with circulation, the national standards of biochemical oxygen demand (BOD5) 27-33 mgL(-1) and chemical oxygen demand (COD) 62-78 mgL(-1) for L. minor and L. gibba, respectively, were reached after treatment. The new results can be used for design calculations regarding expected removal of pollutants by aquatic floating plants.     

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; )     

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)     

Gibberellin and CCC Effects on Flowering and Growth in the Long-day Plant Lemna gibba G3

The application of gibberellic acid (GA₃) to the non-rosette long-day plant Lemna gibba G3 at concentrations from 0.1 to 100 mg/l did not induce flowering on short days and inhibited flowering on long days at concentrations of 1 mg/l and higher. On both short and long days GA₃ concentrations above 1 mg/l caused a decrease in frond size and fresh and dry weight, but an increase in the rate of frond production and thus an increase in the # VF (number of vegetative fronds). Identical results were obtained when gibberellin A₇ was used instead of GA₃.     

Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with ¹³C₆-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR₁) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR₁ has the same amount of DNA per nucleus as the parent line (PL). All jsR₁ cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR₁ at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR₁ had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR₁ and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR₁ had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity.     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

The Effect of Drugs on the Growth of Lemna minor C.

Lemna minor fronds were grown for periods of between 90 and190 days in nutrient solutions to which non-lethal doses ofone of the following drugs were added: sodium nitrite, sodiumazide, brilliant green, proflavine, propamidine isethionate,and dinitrobutyl phenol. Over these periods there was no signof any increase in the growth-rate with time, such as mightbe expected if Lemna showed adaptation to drugs similar to thatfound in micro-organisms. ‘Changeover’ experimentsshowed that the growth-rate of fronds in a drug solution wasgenerally less if they had been previously exposed to that drugthan if they had not. The sensitivity of the fronds, inducedby growth in solutions containing sodium nitrite, persistedover five to six generations of growth in nitrite-free solutions.     

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; )     

Glycolate and glyoxylate stimulation of growth in Lemna gibba

A 10 to 20% stimulation of growth in Lemna gibba L. G3 occurred following the addition of 0.5 to 3 mM glycolate or glyoxylate, although concentrations of 5 mM or higher were inhibitory. Glyoxylate gave a higher stimulation than glycolate. The stimulating effect on growth was obtained in media with or without 2% added sucrose. A higher stimulation was obtained when the plants were cultivated in open flasks in comparison to cultivation in flasks plugged with cellulose stoppers, which presumably retarded gas exchange.     

Effect of abscisic acid on CO2 exchange in Lemna gibba

The effects of abscisic acid (ABA) on photosynthesis, dark respiration, and photorespiration were studied in Lemna gibba L. plants. The initial concentration of ABA in the nutrient solution was 10⁻⁷M and in a few experiments, 10⁻⁶M. The cultures were grown in the same solution for time periods ranging from one hour to 12 days. Net photosynthesis, measured as CO₂ uptake by infrared gas analyser technique, was inhibited after four hours of ABA treatment and reached a minimum after four to seven days depending on the time of the year. After 12 days a substantial recovery of photosynthesis was observed. Dark respiration was significantly stimulated after two to seven days of ABA treatment but then returned to the control level. The transient effects of ABA on photosynthesis and dark respiration corresponded to the previously measured time course of [¹⁴C]-ABA uptake by Lemna . Photorespiration measured as oxygen inhibition of photosynthesis was not affected by ABA.     

Applications of the aquatic higher plant Lemna gibba for ecotoxicological assessment

Although higher plants represent a significant portion of the total biomass in some aquatic environments, their use in ecosystem evaluation has lagged behind that of other organisms. This is partly due to a lack of convenient aquatic higher plant systems that can be employed for ecotoxicological assessment. However, the aquatic C-3 monocot Lemna gibba has many attributes that makes it useful for ecosystem health assessment. In this report, using examples from the literature and our research, some of the applications Lemna has for environmental research are discussed. Toxicant impacts on Lemna can be readily assessed in terms of growth; the plants multiply quickly and changes in biomass (which doubles approximately every 2 days) can be accurately measured by counting leaves. The plants are small, allowing for simultaneous multiple replication. The small size also makes the lighting conditions easy to control; sunlight can be accurately simulated and specific spectral regions can be enhanced or deleted. Lemna is amenable to in vitro chlorophyll and photosynthesis assays, which make excellent companion endpoints for growth. The plants assimilate chemicals directly from the growth medium, facilitating controlled toxicant application. Furthermore, Lemna has a high bioconcentration capacity, indicating a potential for use in bioremediation technologies.