Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = −220 millivolts). In plants maintaining a low pd (−110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H⁺ cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H⁺ -extrusion pump.     

Evaluation of Arsenate- and Vanadate-Associated Changes of Electrical Membrane Potential and Phosphate Transport in Lemna gibba G1

Interference of arsenate and vanadate with phosphate uptakein Lemna gibba L. was studied by measuring voltage changes and(³²P)phosphate uptake. Arsenate proved to be competitive withthe high- and low-affinity phosphate uptake system. It inducedtransient membrane potential changes of up to 120 mV which weresimilar to those induced by phosphate and indicated a cotransportmechanism with at least 2H+/H₂As. The amplitude of the transient arsenate-induced membrane depolarization wasstrongly influenced by phosphate starvation. A permanent membranedepolarization to the diffusion potential was achieved within2 to 6 h in P-starved plants. Thus, arsenate is indeed a stronglycompetitive physiological analogue of phosphate in higher plants. Vanadate was easily transported into L. gibba as concluded fromtransient E_m changes of up to 110 mV. Vanadate interfered onlyslightly and non-specifically with the two phosphate transportmechanisms. Like phosphate, vanadate uptake seems to be an H+-cotransportmechanism, both with similar optima at pH 6.0. Unlike phosphateuptake, vanadate-linked membrane depolarization was not affectedby high intracellular phosphate concentrations. P-starvationdid not enhance the weak long-term effect on E_m. Hence, vanadate,in contrast to arsenate, is not regarded as a physiologicalphosphate analogue. The distinct and rapid vanadate-inducedand permanent membrane depolarization of Avena sativa, Triticumaestivum and Glycine max leaves was not seen in Lemna nor inleaves of Gossypium hirsutum and Nicotiana tabacum. Plasmalemma-enrichedpreparations of L. gibba revealed, however, a high vanadate-sensitiveATPase activity (87%). As a possible explanation for these differencesit is suggested that the latter plant species have cytosolicvanadate-detoxifying properties, i.e. they can reduce vanadateto vanadyl ions, in contrast to the former group of plant species. Key words: Arsenate, vanadate, H⁺/solute cotransport, membrane potential, phosphate competition     

Membrane Potential Changes Related to Active Transport of Glycine in Lemna gibba G1

Accumulation of ¹⁴C-labeled glycine and microelectrode techniques were employed to study glycine transport and the effect of glycine on the membrane potential (Δψ) in Lemna gibba G1. Evidence is presented that two processes, a passive uptake by diffusion and a carrier-mediated uptake, are involved in glycine transport into Lemna cells. At the onset of active glycine uptake the component of Δψ which depended on metabolism was decreased. The depolarized membrane repolarized in the presence of glycine. This glycine-induced depolarization followed a saturation curve with increasing glycine concentration which corresponded to carrier-mediated glycine influx kinetics. The transport of glycine was correlated with the metabolically dependent component of Δψ. It is suggested (a) that the transient change in Δψ reflects the operation of an H⁺-glycine cotransport system driven by an electrochemical H⁺ gradient; and (b) that this system is energized by an active H⁺ extrusion. Therefore the maximum depolarization of the membrane consequently depended on both the rate of glycine uptake and the activity of the proton extrusion pump.     

Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g^-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g^-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (= pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no pH could be measured in the incubation medium, these results can be best explained by a H⁺-hexose cotransport mechanism powered by active H⁺ extrusion at the plasmalemma.Abbreviations LD longday - SD shortday - pd membrane electropotential difference - pd maximum membrane potential depolarization - L light - D dark - FW fresh weight - d days of culture of Lemna gibba - 1X perfusing solution without sugar, see methods     

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)     

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; )     

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.     

A Quantitative Simulation Model for H-Amino Acid Cotransport To Interpret the Effects of Amino Acids on Membrane Potential and Extracellular pH

The H⁺ cotransport of neutral and acidic amino acids induces transient depolarizations of oat coleoptile (Avena sativa L., var Victory) plasma membranes. The depolarizations, which are completed within 1 or 2 minutes, are followed by repolarizations that are nearly completed within another 2 or 3 minutes. Cysteine induced a two-phased alkalinization of the tissue free space during the electrical changes. The first phase was a rapid, linear increase in pH that coincided with the depolarization; the second phase was a slower, also linear, increase in pH that coincided with the repolarization. Reacidification did not occur until cysteine was withdrawn. Five other acidic, basic, and neutral amino acids also induced persistent alkalinization of the free space.

The notable features of these measurements are that free-space pH was measured more directly than previously, that pH changes corresponded in time to the electrical potential changes, and that reacidification of the free space did not occur. The latter observation indicates that net H⁺ efflux did not occur during repolarization and that the repolarizing current was carried by some other ion. We propose that repolarization could have depended upon depolarization-induced changes in passive K⁺ fluxes combined with an enhanced H⁺ extrusion that increased until it equaled, but did not exceed, the enhanced influx of H⁺.

In support of the feasibility of our hypothesis, we present a quantitative simulation model for cotransport. The simulation model also provides an interpretation of the unique electrical effects of histidine and the basic amino acids. In addition, the model focuses attention upon the difficulties of interpreting H⁺-anion cotransport.

     

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.     

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; )     

Spectral dependences of flowering in Lemna perpusilla and L. gibba

Floral induction in Lemna perpusilla and L. gibba was determinedunder continuous irradiation with monochromatic light in spectralranges from 396 to 765 nm. In the former it was induced underwavelengths from about 400 to 550 nm and longer than 700 nm,while in the latter with wavelengths near 400 nm and from about550 to 650 nm. The patterns of these spectral dependences werenearly mirror images and corresponded to the P_fr level in thephotostationary states of phytochrome. (Received December 3, 1974; )     

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; )     

Removal of the sugar inhibition of flowering in Lemna gibba G3 by catecholamines

DL-Epinephrine (10^–8–10^–7 M), DL-norepinephrine(10⁶ M) and DL-isoproterenol (10^–8–10^–6 M)alleviated floral inhibition due to 1% of sucrose, in Lemnagibba G3. The induction period extended by sucrose was curtailedby epinephrine, frond multiplication enhanced by the sugar beingleft unaltered. The pattern of action of catecholamines appearedto be very similar to that of cAMP. DL-Epinephrine, however,was ineffective in the presence of 10^–7 M DL-propranololwhich affected neither flower nor frond production by itself.Quabain and nicotinic acid also nullified the epinephrine actionon duckweed flowering. These and other relevant findings supportthe hypothesis that the cAMP-adenyl cyclase system participatesin the processes of flower induction in this long-day plant. (Received August 21, 1973; )     

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.     

Photosynthesis and nitrogen utilization in exponentially growing nitrogen-limited cultures of Lemna gibba

The photosynthetic performance and nitrogen utilization of Lemna gibba L. G3 adapted to limited nitrogen supply was studied. The plants were adapted to two levels of nitrogen limitation where the nitrogen addition rates were calculated to sustain relative growth rates (RGR) of 0.15 day^?1 and 0.25 day^?1, respectively. The photosynthetic performance of these cultures was compared to nitrogen-sufficient cultures with an average RGR of 0.32 day^?1. Plants transferred from nitrogen-sufficient conditions attained RGR values corresponding to the nitrogen addition rates after 6 to 10 days. Light-saturated net photosynthesis declined during adaptation according to the drop in growth rate, and a concomitant decrease in the respiration rate was recorded. The efficiency of net photosynthesis on a dry weight basis increased with increased nitrogen supply, whereas it was the same in all cultures when expressed on a chlorophyll basis. The light compensation point was unaffected by the nitrogen regime. Limited nitrogen supply resulted in an increased proportion of dry matter in the roots, which led to decreased leaf area ratios. The net assimilation rates also decreased, but not to the same extent as the leaf area ratios. Growth-limiting amounts of nitrogen were added to the cultures once daily, and the net influx of N was higher than the requirement for N, also in adapted cultures with a steady growth rate. This resulted in transient, periodic fluctuations in the NO₃^?, NH₄⁺ and amino acid pools. Also the rates of NO₃^? reduction and NH₄⁺ assimilation fluctuated as did the amino acid assimilation which paralleled NH₄⁺ assimilation. The role of flux rates over the plasmalemma and tonoplast for control of nitrogen assimilation rates are discussed.     

Uptake of Acidic and Basic Sugar Derivatives in Lemna gibba G1

The uptake of acidic and basic sugar derivatives in Lemna gibba L. was studied. Uronic acids applied to the experimental solution (50 millimolar) induced a small decrease of the membrane potential (10 ± 1 millivolt galacturonic acid, and 20 ± 4 millivolt glucuronic acid). After incubation of the plants in a 0.1 millimolar solution of these substrates, no decrease in the concentration of reducing groups in the external solution was detected. Respiration increased by 31% with 50 millimolar galacturonic acid, whereas no effect was found with the same concentration of glucuronic acid. Glucosamine caused a considerable concentration-dependent membrane depolarization. (¹⁴C)glucosamine uptake followed Michaelis-Menten kinetics together with a linear component. Influx of this substrate was inhibited by glucose but the type of competition could not be clearly distinguished. Glucosamine, 50 millimolar, inhibited the respiration rate by 30%. The glucosamine uptake was pH-dependent, with maximum uptake at around pH 7. Lack of enhancement of uptake by low pH as well as the permanent membrane depolarization suggest a uniport mechanism for the charged species of the substrate and an electroneutral diffusion of the uncharged species.     

Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. I. Photosynthesis in vivo

The floating angiosperm Lemna gibba L. was exposed for 2 h to various combinations of photosynthetic photon flux densities and temperature. The extent of photoinhibition of photosynthesis was assayed by measuring the net CO₂ uptake before and after a photoinhibitory treatment, and the time course for photoinhibition was studied. It was found that the maximum quantum yield and the light-saturated rate of CO₂ uptake were affected by the interaction between light and temperature during the photoinhibitory treatment. At a constant photon flux density of 650 μmol m⁻² s⁻¹ the extent of photoinhibition increased with decreasing temperature showing that even a chilling-resistant plant like L. gibba is much more susceptible to photoinhibition at chilling temperatures. About 60% photoinhibition of the quantum yield for CO₂ uptake could be obtained either by a high photon flux density of 1 750 μmol m⁻² s⁻¹ and 25°C or by a moderate photon flux density of 650 μmol m⁻² s⁻¹ and 3°C. The time courses of recovery from 60% photoinhibition produced by either of these two treatments were similar, indicating that the nature of the photoinhibition was intrinsically similar. The extent of photoinhibition was related to the amount of light absorbed in excess to what could be handled by photosynthesis at that temperature. The vital importance of photosynthesis in alleviating photoinhibition is discussed.     

Role of the Transmembrane Potential in the Membrane Proton Leak

The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨ_m) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨ_m (similar to ΔΨ_m in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨ_m. The application of ΔΨ_m up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨ_m and the increase of FA membrane concentration bring about the significant exponential G_m increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA⁻ remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨ_m more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨ_m may be key in mitochondrial respiration and metabolism.