Metabolic responses of Lemna minor to lead ions I. Growth, chlorophyll level and activity of fermentative enzymes

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The growth of Lemna minor was inhibited by lead ions, but the dry to fresh weight ratio increased as the concentration of Pb²⁺ in the medium increased. With increased concentrations of Pb ions, the contents of chlorophyll a and chlorophyll b in roots and fronds were correspondingly lower in comparision with the control. The effect of lead upon activities of some glycolitic and fermentative enzymes in roots of duckweed was examined. The activity of pyruvate kinase decreased with increasing lead concentrations, but cytosolic malate dehydrogenase behaved in an opposite manner. The lowest concentration of Pb stimulated alcohol dehydrogenase; phosphoenolopyruvate carboxylase activity was maintained at relatively constant values at all tested lead concentrations.     

Heavy-metal (Zn,Cd) tolerance in selected clones of duck weed (Lemna minor)

Cryo-microprobe analysis of quench-frozen fronds of a Zn-tolerant clone of Lemna minor exposed to a high level of Zn (300 μM) showed the presence of cellular deposits consisting of Zn, Mg, K and P or Zn, K and P (Zn phytate). The same Zn-tolerant clone of Lemna minor, when exposed to a high level of Cd (30 μM), showed the presence of globular deposits consisting of Cd, K and P in mature fronds, but the immature cells of the enclosed daughter fronds contained relatively large deposits with Cd and S as the main components (Cd-phytochelatin?). Selection for Zn tolerance in a population of Lemna minor was easily achieved but selection for Cd tolerance has so far not been successful. The Zn-tolerant clone also tolerates high levels of phosphate.     

Cadmium Removal by Lemna minor and Spirodela polyrhiza

The present study investigates the ability of two genus of duckweed (Lemna minor and Spirodela polyrhiza) to phytoremediate cadmium from aqueous solution. Duckweed was exposed to six different cadmium concentrations, such as, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L and the experiment was continued for 22 days. Water samples were collected periodically for estimation of residual cadmium content in aqueous solution. At the end of treatment period plant samples were collected and accumulated cadmium content was measured. Cadmium toxicity was observed through relative growth factor and changes in chlorophyll content. Experimental results showed that Lemna minor and Spirodela polyrhiza were capable of removing 42–78% and 52–75% cadmium from media depending upon initial cadmium concentrations. Cadmium was removed following pseudo second order kinetic model. Maximum cadmium accumulation in Lemna minor was 4734.56 mg/kg at 2 mg/L initial cadmium concentration and 7711.00 mg/kg in Spirodela polyrhiza at 3 mg/L initial cadmium concentration at the end of treatment period. Conversely in both cases maximum bio-concentration factor obtained at lowest initial cadmium concentrations, i.e., 0.5 mg/L, were 3295.61 and 4752.00 for Lemna minor and Spirodela polyrhiza respectively. The present study revealed that both Lemna minor and Spirodela polyrhiza was potential cadmium accumulator.     

Investigations on Cytokinins. I. Effect of 6-Benzylaminopurine on Growth and Starch Content of Lemna minor

An excessive starch accumulation in the fronds of Lemna minor L. was observed microscopically and confirmed by direct starch determinations after application of growth inhibiting 6-benzylaminopurine concentrations. As a high growth rate was coupled with a low starch content and vice versa, the influence of the cytokinin on growth and starch content was compared. It appeared that apart from the expected influence of the growth rate on the starch content, 6-benzylaminopurine favoured starch accumulation also by itself.     

Ecology of SO2 resistance: I. Effects of fumigations on gas exchange of deciduous and evergreen shrubs

Summary A unique gas exchange system is described in which photosynthesis, transpiration, and stomatal conductance can be measured on leaves during SO₂ fumigations. SO₂ concentrations can be continuously monitored and manipulated between 0 and 2.0 ppm. Rates of total SO₂ uptake and SO₂ absorption through stomates of a fumigated leaf can also be determined.Using this system we compared the effects of SO₂ on the gas exchange rates of two shrub species that co-occur in the Califormian chaparral. Diplacus aurantiacus, a deciduous shrub, was more sensitive to SO₂ fumigation than Heteromeles arbutifolia, an evergreen shrub. The differences in photosynthetic sensitivity could be attributed, in large part, to differential SO₂ absorption rates.     

ABSCISIC ACID EFFECTS ON FRONDS AND ROOTS OF LEMNA MINOR L.

Plants of Lemna minor L. were grown in axenic culture in order to investigate the direct effect of abscisic acid (ABA) on individual fronds and roots. Both frond and root growth rates were inhibited by 60%, and total growth of both organs was reduced by 30% with ABA concentrations of 2 mg/l. Abscisic acid inhibited frond reproduction, and treated fronds tended to remain attached to parents. Reduction of root elongation and frond expansion occurred within 1–4 hr. Prolonged exposure to ABA inhibited both cell enlargement and cell division in the roots. The results are discussed in the light of current views on the control of growth with endogenous levels of promoter and inhibitor hormones.     

The accumulation of amino-acids and humic substances in media conditioned by axenic and non-axenic duckweed (Lemna minor L.) and their possible ecological significance

The concentrations of total free amino acids (TFAA) and humic substances (HS) accumulating in media conditioned by axenic and non-axenic duckweed fronds (Lemna minor L.) were analyzed at various time intervals over a 21-day incubation period with the aid of a Shimadzu HPLC system. In the non-axenic Lemna cultures, the concentrations of both TFAA and HS continued to increase throughout the incubation period, although the rate of increase was higher in the initial stages. In contrast, the concentrations of both TFAA and HS reached asymptotic values in media conditioned by non-axenic Lemna after 10–12 days. As a result, the concentrations of both FAA and HS became significantly higher in media conditioned by axenic Lemna fronds than in those conditioned by non-axenic Lemna from days 10–12 until the end of the experiment. The possible reasons for the differences in the accumulation patterns of TFAA and HS in media conditioned by axenic and non-axenic Lemna and their ecological significance are discussed.     

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.     

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

An experimental set-up which enabled non-invasive, real-time reactive oxygen species (ROS) visualization on a whole plant level was constructed. In the test organism, Lemna minor L. (common duckweed), apoplastic and symplastic oxidative stress was evaluated by exposure to menadione (50 μM), menadione (50 μM) + ascorbate (100 μM) or neither for control. Menadione (50 μM) caused a statistically significant increase in H₂DCFDA fluorescence in the apoplast after 60 minutes of exposure. The addition of ascorbate (100 μM) in the test medium significantly decreased apoplastic oxidative stress. 50 μM menadione caused an increase in symplastic H₂DCFDA fluorescence in 57% of fronds. The exposure of L. minor plants to both menadione and ascorbate decreased the rate of fluorescence intensity accumulation in the symplast to control levels. The method has proven to be quick and straightforward and could be applied to a range of chemicals in various physiological and toxicological plant studies. The advantages of the set-up and different possible artefacts are discussed.     

The effect of deuterium oxide on protein turnover in Lemna minor

Lemna minor fronds transferred to a sterile culture medium containing 50% (v/v) deuterium oxide (²H₂O) rapidly undergo a loss of soluble protein with a corresponding increase in free amino acids. The loss of protein is due to two factors: (i) the inhibition of protein synthesis for 4 h followed by a slower rate of synthesis than normal, (ii) a rapid 9–10 fold increase in protein degradation. In plants grown for longer periods (3–6 days) in 50% ²H₂O medium, protein synthesis is inhibited by 20% and the rate constant of degradation is 2–3 times that measured in fronds growing in normal (H₂O containing) complete medium. The initial loss of protein is not due to the breakdown of any specific protein fraction. Investigation of several enzymes indicates that all proteins are catabolised in response to ²H₂O treatment. The implications of these results with regard to the interpretation of density-labelling experiments are discussed.     

High SO2 resistance ofClethra barbinervis established in a smoke-polluted area of Ashio,Tochigi Prefecture,Japan

The effect of SO₂ on the photosynthesis ofClethra barbinervis collected from a smoke-polluted area near the Ashio copper smelter in Tochigi Prefecture was compared withC. barbinervis collected from a nonpolluted district in Tsukuba, Ibaraki Prefecture andQuercus mongolica var.grosseserrata grown in a nonpolluted field in Nagano Prefecture. The plants were exposed to 0.5–1.5 p.p.m. SO₂ for 90 min (short-term) and to 0.3 p.p.m. SO₂ for 31–39 days (long-term). TheClethra plants from both sites had a lower intrinsic stomatal conductance and photosynthetic rate thanQuercus plants. Short-and long-term fumigation caused stomatal closure inQuercus plants, but had little effect on the stomatal conductance ofClethra plants. Under short-term fumigation, nonstomatal photosynthetic inhibition per unit of absorbed SO₂ was smallest inClethra plants from Ashio. Long-term fumigation caused photosynthetic decline and visible foliar injury toQuercus plants, but had no effect onClethra plants from Ashio. Consequently,Clethra plants from Ashio had a higher photosynthetic rate thanQuercus plants after long-term fumigation. These results suggest thatC. barbinervis populations in the smoke-polluted area of Ashio had evolved high SO₂ resistance connected with SO₂ detoxification ability in mesophyll cells.     

Changes in Proline Content Accompanying the Uptake of Zinc and Copper by Lemna minor

Lemna minor L. grown in Hoagland solution containing zinc (10ppm) or copper (5 ppm) for 4 d accumulated high levels of thesemetals. Zinc and Cu accumulation in the test plants was accompaniedby a specific pattern of change in proline content. The lattershowed a steep rise during early stages (peak at 12-24 h) followedby a gradual decline until 96 h of treatment. In a dose-responsestudy, lower metal concentrations induced a sharp rise in prolinelevel with a maximum value at 5 ppm, which declined when theconcentration was further enhanced. There was a correspondencebetween the level of proline and total free amino acids in metaltreated plants. The possibility of proline involvement in tolerancemechanisms to heavy metals has been discussed.Copyright 1993,1999 Academic Press Heavy metals, zinc, copper, proline, total free amino acids, Lemna minor     

Arsenic Uptake by Lemna minor in Hydroponic System

Arsenic is hazardous and causes several ill effects on human beings. Phytoremediation is the use of aquatic plants for the removal of toxic pollutants from external media. In the present research work, the removal efficiency as well as the arsenic uptake capacity of duckweed Lemna minor has been studied. Arsenic concentration in water samples and plant biomass were determined by AAS. The relative growth factor of Lemna minor was determined. The duckweed had potential to remove as well as uptake arsenic from the aqueous medium. Maximum removal of more than 70% arsenic was achieved at initial concentration of 0.5 mg/l arsenic on 15th day of experimental period of 22 days. Removal percentage was found to decrease with the increase in initial concentration. From BCF value, Lemna minor was found to be a hyperaccumulator of arsenic at initial concentration of 0.5 mg/L, such that accumulation decreased with increase in initial arsenic concentration.     

An in vivo analysis of the effect of SO2 fumigation on photosynthesis in Zea mays.

Fumigation of leaves with SO₂ can reduce the capacity for photosynthetic CO₂ uptake even in the absence of visible symptoms of damage. In vitro studies suggest that this invisible injury to intact leaves could be affected by damage to each of the main stages in the photosynthetic process. Reduced stomatal apertures may also reduce photosynthesis following SO₂ fumigation. The responses of CO₂ uptake by leaves to intercellular CO₂ concentration and to absorbed light provide information for quantitative separation of the in vivo contribution of the different stages of photosynthesis to reduction in overall rate. This study uses these techniques to examine the basis of reduction in CO₂ uptake in Zea mays cv. LG11 leaves following short-term fumigation with SO₂. Fumigation with 33 μmol m^–3 SO₂ for 30 min reduced light saturated CO₂ uptake by about one-third. An even greater reduction in light limited CO₂ uptake was observed and with no significant change in light absorptance this was attributed to a reduced quantum yield of photosynthesis. The light saturated CO₂ uptake rate and the stomatal conductance decreased in parallel. However, the relationship of CO₂ uptake to the intercellular CO₂ concentration suggested that the reduced stomatal conductance did not account for the reduced rate of CO₂ uptake following fumigation. Both the initial slope and plateau of this relationship were significantly reduced, suggesting that both carboxylation efficiency and capacity for regeneration of CO₂ acceptor were diminished by SO₂ fumigation. The operating intercellular CO₂ concentration indicated that both processes were co-limiting, before and after fumigation. The time required for induction of photosynthetic CO₂ uptake on illumination was approximately doubled following SO₂ fumigation, showing that fumigation impairs the ability of the photosynthetic apparatus to adapt to fluctuations in light level.     

Antioxidant systems in the leaf apoplast compartment of Pinus pinaster Ait. and Pinus radiata D. Don. Plants exposed to SO2

The activities of guaiacol peroxidase (GuPOD), ascorbate peroxidase (ASAp), superoxide dismutase (SOD) and ascorbate/glutathione cycle (AGC) enzymes, together with ascorbate (ASC) and glutathione contents, were determined in apoplastic-fluid and cell-wall fractions of needles of Pinus pinaster Ait. and Pinus radiata D. Don. exposed for up to 6 months to SO₂ (0.01 ppm or 0.30 ppm) in fumigation chambers. AGC enzyme activities (monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) were in all cases undetectable, as was glutathione content. In needles of P. pinaster plants exposed to SO₂, ascorbate content and all enzyme activities considered (except AGC enzymes) increased. The increases were most marked in response to the higher SO₂ concentration. In needles of P. radiata, similar but less marked responses were observed. These findings suggest a) that enzyme activities and ascorbate contents increase in order to deal with the reactive oxygen intermediates produced during long-term contamination with SO₂, and b) that P. pinaster has more effective defences against contamination of this type than P. radiata.     

Effect of abscisic acid on photosynthetic products of Lemna minor

Lemna minor fronds were grown on nutrient only, or nutrient plus 10^?6M abscisic acid (ABA) for 2 or 8 days. After various ¹⁴CO₂ pulse-chase time periods, the fronds were harvested and the photosynthetic products separated into acidic, lipid, residue, sugar and amino acid fractions. Compared with the control fronds, total ¹⁴C-fixation was 15% higher in the 2 day ABA-treatment and 6% lower in the 8 day ABA-treatment. This pattern was reflected in all the fractions examined, and it appeared that ABA did not alter the distribution of ¹⁴C between the photosynthetic products during the ¹⁴CO₂ pulse. During the chase, less ¹⁴C was lost from the carbohydrate fractions in the ABA-treated fronds than in the control fronds. The results indicate that the previously reported ABA-mediated increase in carbohydrate levels was a consequence of decreased degradation rather than an increase in synthesis from assimilated carbon.     

The Use of Chemical and Infra-red Spectral Analyses to Verify the Identification of Two Species of Duckweed, Lemna minor L. and Lemna gibba L. Grown Under Defined Conditions

Flat forms of the fronds of Lemna gibba L., used widely in laboratoryexperiments, can be easily confused with Lemna minor Chemicalanalyses of the two species, grown under defined laboratoryconditions, showed clear distinctions between the two speciesin relation to total protein content and the content of individualmonosaccharides. Infra-red spectral analysis confirmed the chemicaldata for protein content and also revealed differences in thetypes of proteins present in the two species. Chemical and infra-redspectral analyses, particularly the amide I: carbohydrate absorbanceratio, provide a rapid and reliable means of verifying betweenthe two species, which are often difficult to identify withcertainty from morphological differences. Key words: Gibbosity, infra-red spectroscopy, Lemna gibba, Lemna minor, monosaccharides, proteins     

Short term exposure of Lemna minor and Lemna gibba to mercury, cadmium and chromium

The effects of mercury (Hg), cadmium (Cd) and chromium (Cr) in concentrations ranging from 0.02 to 20 mg L^?1 applied for 24 h were assessed in Lemna minor and Lemna gibba by measuring changes in protein concentration, ascorbic acid, phenolics, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), the activity of guaiacol peroxidase (G-POX) and catalase (CAT). Ascorbic acid, phenolics, catalase and guaiacol peroxidase played a key role in the antioxidative response of L. gibba. Inadequate activity of antioxidant enzymes in the L. minor resulted in MDA and H₂O₂ accumulation. In both used species, Hg treatment decreased protein content and increased CAT and G-POX activity, but decreased MDA and H₂O₂ levels. Cadmium and chromium had opposite impacts on two used Lemna species on almost all observed parameters. Enhanced antioxidative responses of L. gibba to lower concentrations of Hg, Cd and Cr indicated greater abiotic stress tolerance than L. minor.     

Effect of Cyclic Phytoremediation with Different Wetland Plants on Municipal Wastewater

Phytoremediation is a promising cleanup technology for contaminated soils, groundwater, and wastewater that is both low-tech and low-cost. The objective of this study was to investigate the ameliorative effect of phytoremediation on municipal wastewater (MWW). For this purpose, a phytoremediation garden was established using different aquatic plants species [Pistia stratiotes, Eichhornia crassipess, Hydrocotyle umbellatta, Lemna minor, Tyhpa latifolia, and Scirpus acutus ] in seven earthen pond systems (P1-P7) for the cyclic treatment of MWW. The physico-chemical analysis of MWW was carried out before and after the cyclic phytoremediation. Results showed that pH, EC and turbidity of MWW were reduced by 5.5%, 33.7%, and 93.1%, respectively after treatment (from P1 to P7). Treatment system also reduced total dissolved solids (TDS) by 35.2%, Cl by 61%, HCO₃ by 29.2%, hardness by 45.7%, Ca by 32.3% and Mg by 55.9%. Nitrate concentration was reduced by 77.6% but SO₄ was enhanced slightly. An ameliorative combined effect of wetland plants namely L. minor, T. latifolia, and S. acutus on MWW was noticed. Sequential phytoremediation with a mixture of plants was more effective than that relying only on a single plant species.     

Leaf surface properties of Norway spruce needles exposed to sulphur dioxide and ozone in an open-air fumigation system at Liphook

Samples of current-year and 1-year-old foliage were taken from Norway spruce (Picea abies (L.) Karst.) trees in April 1991, 4 months after a 3–4 year controlled fumigation with O₃ and SO₂ in the open at Liphook, south-east England. Trees were grown in seven plots, and treated in a factorial design with three levels of SO₂ and two levels of O₃ (ambient and c. 1.3 × ambient), with an extra ambient air plot. All statistical analyses were made on plot means. Leaf wettability, as measured by the contact angle of water droplets, was significantly affected by needle age and by SO₂ treatment (P≤0–05. in older needles, decreasing with increasing SO₂ concentration. There was no effect of O₃ on wettability, and no effect of any treatment on amounts of surface wax extracted by immersion of needles in chloroform. Electrolyte leakage rates from detached current-year needles were not affected by prior exposure to O₃, but decreased significantly (P= 0.034) with increasing exposure to SO₂. There was no detectable effect of fumigation on the rate of water loss from detached needles. Similarly, there was no effect of fumigation on the dry weight/fresh weight ratio of needles. The total sulphur content of needles increased significantly (P≤0.0001) with exposure to SO₂ and with needle age. Amounts of water-extractable sulphate, however, varied greatly among plots, but with no pattern with respect to fumigation treatment. It is concluded that leaf wettability and electrolyte leakage rates may be good indicators of the persistent effects of SO₂ on Norway spruce growing in the open air, and that the observed changes in leaf surface properties in response to SO₂ fumigation have implications for the processes, both biotic and abiotic, that occur on leaf surfaces.