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.     

Effects of artificial ultraviolet-B radiation on growth and fatty acid composition of duckweed (Lemna minor)

1. Duckweed (Lemna minor), collected either in summer or early fall was exposed under laboratory conditions to control (photosynthetically active and UV‐A radiation) or experimental (control plus UV‐B radiation) conditions. 2. Growth and survival were determined by counting the number of green, and brown/white fronds following 1–5 or 11 days of irradiation. Growth of duckweed was impaired by exposure to UV‐B radiation in the fall experiment but not in the summer. 3. Fatty acid compositions were analysed following 5 or 11 days of irradiation and a recovery period of 0, 5, 29 or 40 h. Concentrations of the major fatty acids, palmitic, linoleic (LA) and α‐linolenic (ALA) acids were similar in the summer and fall duckweed collections, but the summer samples had higher concentrations of the desaturation products of LA and ALA. 4. UV‐B exposure had small, but significant, and contrasting effects on duckweed fatty acid concentrations. In the summer experiment, duckweed exposed to UV‐B had slightly lower concentrations of major fatty acids than control duckweed, while the reverse was true in the fall experiments. 5. These minor effects of UV‐B on concentrations of LA and ALA would be unlikely to have a major impact on the supply of these essential fatty acids from duckweed to freshwater food webs.     

Regulation of the xanthophyll cycle pool size in duckweed (Lemna minor) plants

Duckweed ( Lemna minor L.) plants grown under high light are characterized, when compared to low light acclimated plants, by a higher xanthophyll cycle (VAZ) pool content, but also by a higher proportion of photoconvertible violaxanthin and a superior ability to synthesize VAZ pigments. When duckweed plants were transferred to a high light environment a general response was the quick adjustment of the carotenoid composition, mainly xanthophyll cycle pigments. These changes resulted from a balance between a process of continuous light-independent carotenoid degradation and a light-induced accumulation. The use of norflurazon, an inhibitor of carotenogenesis, allowed us to demonstrate that the observed light induced increase of the VAZ pool was mainly caused by de novo synthesis through carotenogenesis. The extent of light-induced carotenogenesis was proportional to the light treatment and also to the operation of the VAZ cycle since it was partly abolished by treatments leading to a low activity of the VAZ cycle, such as low light, DTT or DCMU. These results suggest that not only the light itself, but also a mechanism triggered by a factor associated with the de-epoxidation state of the VAZ cycle controls carotenogenesis at some point before phytoene formation in the terpenoid biosynthesis pathway.     

The distribution of lead in duckweed (Lemna minor L.) root tip

While considerable information on lead distribution in the cells of terrestrial plants has been collected, little is known about lead localization in the cells of the aquatic plant. Lemna minor L. (duckweed) roots were examined using X-ray microanalysis. After 1-h treatment with lead, its concentration was the highest in small vacuoles. After 6 and 12 h, the lead content of cell walls gradually increased. The changes of lead level between vacuoles and cell walls may result from redistribution of this metal from symplast to cell walls or it may reflect increased apoplastic transport. Lead was not found in the ground cytoplasm of any variants of the experiments. This fact and presence of lead in small vesicles suggests that endocytosis may play the role in lead uptake in Lemna.     

The effect of pH on the population growth of three species of duckweed: Spirodela oligorrhiza, Lemna minor and Wolffia arrhiza

Three species of duckweed, Spirodela oligorrhiza, Lemna minor and Wolffia arrhiza were grown under aseptic conditions on both buffered and unbuffered solutions of Jacob's media. Media with manually regulated pH levels were also used. Growth on unbuffered media is initially rapid but eventually inhibited, probably by increased pH levels. On buffered media growth is poor and effects of buffers cannot be separated out. These media give inadequate pictures of the species’ responses to changes in pH. Growth is most successful on media with regulated pH where sustained logarithmic population increases were achieved. Spirodela and Lemna rates are symmetrical about an almost neutral, optimal pH, declining fairly rapidly away from the optimum. Wolffia has an optimum at pH 5 and growth declined with increasing pH. All three species have optima at, or below, the neutral point. The range of tolerance of duckweeds is broader than has previously been suspected. Estimated lower limits, optimum and upper limits for each species are: Wolffia, pH 4·5–0·10, Lemna pH 4–6·2–10, Spirodela pH 3·7–0·10. Growth rate along a pH gradient is best described by means of polynomial equations: second-degree equations are sufficient for Spirodela and Lemna but a fifth-degree equation is required for Wolffia. Rates of population growth are similar for all species. In decreasing order they are: Wolffia, Lemna, Spirodela. However, in biomass units Lemna grew more than six and Spirodela seventeen times faster than Wolffia.     

A multigenerational effect of parental age on offspring size but not fitness in common duckweed (Lemna minor)

Classic theories on the evolution of senescence make the simplifying assumption that all offspring are of equal quality, so that demographic senescence only manifests through declining rates of survival or fecundity. However, there is now evidence that, in addition to declining rates of survival and fecundity, many organisms are subject to age‐related declines in the quality of offspring produced (i.e. parental age effects). Recent modelling approaches allow for the incorporation of parental age effects into classic demographic analyses, assuming that such effects are limited to a single generation. Does this ‘single‐generation’ assumption hold? To find out, we conducted a laboratory study with the aquatic plant Lemna minor, a species for which parental age effects have been demonstrated previously. We compared the size and fitness of 423 laboratory‐cultured plants (asexually derived ramets) representing various birth orders, and ancestral ‘birth‐order genealogies’. We found that offspring size and fitness both declined with increasing ‘immediate’ birth order (i.e. birth order with respect to the immediate parent), but only offspring size was affected by ancestral birth order. Thus, the assumption that parental age effects on offspring fitness are limited to a single generation does in fact hold for L. minor. This result will guide theorists aiming to refine and generalize modelling approaches that incorporate parental age effects into evolutionary theory on senescence.     

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.     

Diatom ecology in the phyllosphere of the common duckweed (Lemna minor L.)

Epiphyton associated with thick, floating mats of the common duckweed (Lemna minor L.) was studied at four sites in western Canada between 1985 and 1988. Maximum epiphyton abundance generally occurred in spring as biomass of the duckweed mat was increasing. Epiphytic biomass was low during summer and increased at some sites in autumn with mat decomposition. The community was composed mostly of diatoms and, during summer, photosynthetic bacteria. Species richness of the diatom flora was low, suggesting that duckweed mats are environments to which few species are adapted. Photosynthesis - irradiance curves indicated that duckweed epiphyton was not adapted to low light levels that occurred in the mat (< 1 % of ambient), suggesting they may survive via other means of nutrition. The mat phyllosphere was also characterized by wide spatial and temporal variation in temperature, and sharp vertical profiles of dissolved oxygen and nutrients.     

Phytomonitoring of pulverized fuel ash leachates by the duckweed Lemna minor

The duckweed Lemna minor is one of the smallest vascular plants with a known strong capacity for metal accumulation. L. minor is proposed as a phytomonitor for coal ash drainage systems and for bio-assay studies directed to complexation and speciation. The duration of the experiment can be restricted to fourteen days; it is then possible to determine accurate data of differences in growth of the clone forming plant by using image processing techniques. Leaching of pulverized fuel ash (PFA) with acetic acid according to EPA instruction resulted in effects attributed to the acetic acid itself rather than to the metals in solution. Toxic effects of both leachates, natural and artificial, are discussed. The order of toxicity of metals studied so far in separate metal experiments is Cd > Cu > Zn > As(Arsenite) > Se(Selenite) > Ge > B > Mo.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

General characteristics of normal and stress-enhanced protein degradation in Lemna minor (duckweed).

The general features of protein degradation in Lemna minor were studied by using a double-isotope technique. In common with several animal systems, there are correlations between the relative rate of protein degradation on the one hand and molecular weight, charge and carbohydrate content on the other. Large proteins, acidic proteins and non-glycosylated proteins are degraded relatively more rapidly than small or basic proteins, or glycoproteins. The correlations with size and carbohydrate content are explicable on the basis of differential susceptibility to Pronase, whereas the charge correlation cannot be explained on the basis. In addition, acidic proteins are not generally of higher molecular weight than neutral or basic proteins. The correlations are found in fronds growing in normal complete medium and in fronds transferred to medium lacking nitrate of made 50% (v/v) with respect to deuterium oxide, both of which are conditions that induce a large increase in protein breakdown in Lemna. Thus basal protein degradation and enhanced degradation do not appear to differ fundamentally in their general characteristics. The results are discussed in relation to the reported features of normal and enhanced proteolysis in animal tissues and to the possible mechanism of protein degradation in Lemna.     

Photosynthesis in ozone-exposed duckweed (Lemna gibba)

The photosynthetic light saturation curve in duckweed was lowered by 20–25% after ozone exposure (300 nmol mol⁻¹, 1 h). The light flux and oxygen concentration during ozone-exposure had no effect on reduction of net photosynthesis. Net photosynthesis and photorespiration were both depressed by about 40% after exposure for 1 h to 360 nmol mol⁻¹ ozone. We could not find any change in dark respiration after ozone exposure below 300 nmol mol⁻¹. When the concentration of ozone was doubled from 150 nmol mol⁻¹ to 300 nmol mol⁻¹, the uptake of ozone in duckweed changed from 100 nmol m⁻² s⁻¹ to 170 nmol m⁻² s⁻¹. We found no differences in fluorescence (pattern) between ozone treated plants and the control plants during a period of 150 min after ozone treatment, but there was an increase in synthesis of the Dl-protein and a significant reduction in degradation after ozone treatment (300 nmol mol⁻¹, 1 h). These results, together with fluorescence measurements, indicate that photochemical electron transport was not responsible for the ozone-induced reduction in net photosynthesis.     

The influence of sublethal concentrations of sulfur dioxide on morphology,growth and product yield of the duckweed Lemna minor L.

Summary There was no disturbance in the growth of Lemna minor L. with a SO₂ concentration of up to 0.3 ppm in air. A SO₂ concentration of 0.6 ppm caused an initial depression of the growth rate of about 25%, but in the course of adaptation, the rate rose to the values of the control. The average dry weight per frond was not influenced by the SO₂ fumigation. The initial sporadic appearance of chloroses by fumigation with 0.6 ppm SO₂ was considered a sign of the proximate toxicity limit for Lemna minor L. With 0.15 ppm SO₂ in air, the size of the fronds was reduced. The average surface of the fronds was diminished by 0.3 ppm SO₂ for about 16% as compared with the control plants.The protein remained quantitatively uneffected up to a SO₂ concentration of 0.6 ppm. As a qualitative influence of SO₂, the nitrogen content of the proteins remained constant, but the sulfur content of the proteins increased.Under 0.3 and 0.6 ppm SO₂, the starch content decreased immediately by 20–30%, under 0.15 ppm SO₂ the decrease reached the same level after a longer time than in the case of the higher concentrations.The SO₂ concentrations up to 0.6 ppm had no effect on chlorophyll concentration.The contents of C, N, H, P, K, Na, Ca, Mg, Mn, and Fe were not effected by SO₂ fumigation.Conclusion: SO₂ may have some effects on product yield, even under low concentrations, without provoking acute damage; the plant is able to adapt by regulation of its metabolism, and enters a new steady state.The study was supported by a grant of the Swiss National Science Foundation (project number 3.866.71)     

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 occurrence of apiose in Lemna (duckweed) and other angiosperms

1. The branched-chain pentose apiose reacts with the benzidine–trichloroacetic acid reagent on paper chromatograms to give a yellow spot with·an intense white fluorescence in ultraviolet light; on chromatograms developed with butanol–acetic acid–water this spot lies between fucose and rhamnose. 2. Examination of paper chromatograms of hydrolysates of whole plant material has shown the presence of a substance with these properties in a wide variety of species. 3. Among the plants examined two members of the Lemnaceae (Lemna minor and Wolffia arrhiza) were found to be especially rich sources, comparable with Posidonia australis (Bell, Hardwick, Isherwood & Cahn, 1954). 4. Measurements of the apiose content of fractions derived from Lemna have shown that the sugar is present at a concentration of about 4% in the holocellulose, and part of this is retained in the α-cellulose left by extraction with 24% (w/v) potassium hydroxide containing borate.     

The effect of nitrogen deficiency on the growth and metabolism of Lemna minor L.

When Lemna is deprived of nitrogen, growth and respiration decrease and the pattern of ¹⁴CO₂ release from [1-¹⁴C]glucose and [6-¹⁴C]glucose is consistent with a relatively strong inhibition of glycolysis. Protein degradation is enhanced but the concentration of free amino acid decreases. It is argued that the biological significance of the increased protein degradation does not lie in its contribution to respiration but as a mechanism to replace one set of enzymes adapted to a particular environmental condition (high nitrogen) with another set of enzymes adapted for low nitrogen in the environment. The change in enzyme pattern associated with the change from high to zero nitrogen in the growth medium has been examined for nine enzymes. The changes in activity observed are consistent with the observed apparent inhibition of glycolysis during nitrogen starvation, but do not explain the inhibition of the pentose phosphate pathway.     

Importance of dissolved organic matter produced by duckweed (Lemna minor) in a southern English river

SUMMARY. 1. The quantity of dissolved organic carbon (DOC) released by axenic Lemna minor cultures in the laboratory has been determined by an absolute method and by ¹⁴C labelling.
2. Good agreement was obtained between the two methods and from 1.1% to 2.6% of the total carbon fixed was released.
3. The DOC produced by L. minor has been analysed by ultrafiltra- tion and compared with a similar analysis of DOC in natural river water.
4. The results of ullrafiltration analyses indicate that the proportion of low molecular weight (<1000 Daltons) material in DOM produced by axenic L. minor is significantly greater than is found in its natural habitat.