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.     

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.     

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.     

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.     

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 xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana

Arabidopsis plants overexpressing beta-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.     

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.     

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.     

The effect of various aquatic bacteria on the growth and senescence of duckweed (Lemna minor)

Five different species of freshwater bacteria ( Pseudomonas sp., Vibrio sp., Klebsiella sp., Enterobacter sp., Serratia sp.) and a mixed natural population were used separately to inoculate cultures of axenic duckweed ( Lemna minor ). Inoculation with Vibrio sp. caused the final population density of Lemna plants to be significantly greater after 52 d than that of either axenic controls or Lemna inoculated with a mixed bacterial community. Inoculation with Pseudomonas sp. caused the final population density of Lemna to be significantly higher than with the mixed bacterial treatment. Inoculation of Lemna with Klebsiella sp., Enterobacter sp. or Serratia sp. resulted in higher plant populations compared with controls, but these differences were not statistically significant. The presence of a mixed community of bacteria did not significantly affect the final population density of Lemna compared with the controls. However, Lemna plants inoculated with a natural population of bacteria showed significantly higher levels of senescence compared with the other five treatments and the controls. None of the five single bacterial taxa used appeared to have any significant effect of the sensescence of duckweed.     

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.     

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.     

Engineering triacylglycerol accumulation in duckweed (Lemna japonica)

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1- to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 μm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.     

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.     

Sensitivity of duckweed (Lemna major) to ultraviolet-B radiation

The sensitivity of an important aquatic macrophyte, duckweed (Lemna major), to UV-B radiation was studied under experimental conditions at three different doses designated as no, mild, and severe injury dose by observing visible injury symptoms and estimating levels of chlorophyll, pheophytin, carotenoids, protein, starch, free sugar, and peroxidase activity. Laboratory-grown duckweed plants were exposed to UV-B radiation at 0.4 mW/cm(2) intensity for different time periods. Mild and severe injury were developed at 6.48 and 8.64 J, respectively. Peroxidase activity increased at all the exposure levels. Dose-dependent decrease in chlorophyll and starch with drastic depletion in protein and free sugar content were observed. Pheophytin and carotenoids content increased at no injury level, but decreased at higher exposure level. The results indicate that ambient UV-B radiation at the indicated level acts as a physiological stress in Lemna major.     

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.     

Regulation of root growth by gibberellin in Lemna minor

Hormonal control of root growth was studied in Lemna minor. Although addition of gibberellic acid (GA3) to the culture medium did not promote the root growth, a gibberellin biosynthesis inhibitor, uniconazole P (Un-P), significantly inhibited root growth. Both length and diameter of roots in Un-P-treated plants were significantly smaller than those in control plants, mainly caused by inhibition of cell division. In epidermal cells, the length was slightly decreased and the width increased by Un-P treatment, indicating inhibition of elongation growth. GA3 completely nullified the inhibition caused by Un-P. Transverse cortical microtubules (CMTs) of epidermal cells in the elongation zone were significantly fragmented by treatment with Un-P, but not by that in the presence of GA3. The cellulose microfibril array in the Un-P-treated cells was more random and more oblique than that in the control cells. These results suggested that root growth in L. minor is regulated by endogenous gibberellin.