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.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3.I. Effects of nitrogen Limitation

The growth rate of Lemna giba L. G3 was varied by limiting the supply of nitrogen (N) under otherwise constant condition. Two experimental approaches were used. 1) A series of suboptimally growing cultures were supplid daily with exponentially increasing doses on N. 2) Optimally growing cultures were transferred to a N-fre medium and cultivated in it for 10 days. Lveles of starch, sucrose, glucose, fructose, and systems. At RGR ranging from optimal to 50% of optimal caused decreased levels of soluble sugars, but increased lvels of starch.Starch accumulation showed a strong negative correlation with the CO² assimilation rate, indicating increased triose phosphate/inorganic phosphate (TP/Pⁱ) ratio in the chloroplast causing end product inhibition of photosyntheisis. The data indicat the quantitative rlationship betwen the photosynthetic activity and the carbon utilization rate influnces the activity of the sucrose synthesis pathway and thus the rate of the triose hosphate/Pⁱ exchange at the chloroplast membrane, which in turn regulates the activity of starch synthesis and the Calvin cycle.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.     

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.     

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.     

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.     

pH及无机氮化合物对细脉浮萍生长的影响

研究以细脉浮萍 ( Lemna aequinoctialis)为研究对象 ,在完全培养液的基础上模拟生活污水的氨氮、硝酸氮浓度和 p H范围 ,研究了培养条件对其生长的影响。结果表明 ,这个浮萍种对 p H耐受的低限在 4～ 5之间 ,最适 p H范围为 5～ 6。非离子态的氨会对其生长产生抑制作用 ,在氨氮为唯一氮源的条件下 ,非离子态氨的浓度大于 0 .1 mg/L时会对此种的生长产生明显的抑制作用 ,接近 2 mg/L时 ,基本上致死。在研究的氨氮和硝酸氮浓度范围内 ( <40 mg N /L) ,铵根离子和硝酸根离子浓度增加可促进细脉浮萍的生长。氨氮中生长的植株过氧化物酶活性高于硝酸氮中的活性。     

A combination method based on chitosan adsorption and duckweed (Lemna gibba L.) phytoremediation for boron (B) removal from drinking water

The metalloid boron (B) and its compounds widely exist in the environment, and boron can have hazardous effects on plants, animals, and human beings when it is found in high concentrations in water bodies. It is difficult and costly to remove B with conventional treatment methods from drinking water. Therefore, alternative and cost-effective treatment techniques are necessary. In this study, for the first time, a novel and environmentally friendly method based on the phytoremediation ability of chitosan and duckweed (Lemna gibba L.) combination was evaluated for B removal from drinking water. Our results from batch adsorption experiment indicated that the highest B uptake capacity of chitosan bead was found as 3.18 mg/g, and we determined the optimal B sorption occurs at pH value of 7. The Langmuir isotherm and pseudo-second-order kinetic model better fitted the equilibrium obtained for B removal. B in drinking water could be reduced to less than 2.4 mg L ^?1 when 0.05 g of plant-based chitosan beads and 12 L. gibba fronds were used in the 4-day treatment period.     

Nodular somatic embryogenesis and frond regeneration in duckweed,Lemna gibba G3

Duckweed(Lemna gibba) is a useful model system for elucidating plant development, but the techniques needed for regenerating fronds from calli are not yet well established. This study examined the effects of auxin, sucrose, and gelling agents on callus and frond formation inL. gibba G3. After three weeks of culturing on a solid medium, two types of calli were observed: watery, pale-green, and undifferentiated; or white, compact calli that were organized into nodules and which resembled somatic embryogenie calli. Homogeneous callus lines were produced through selective subculture. To induce nodular calli, auxin (2,4-D) was absolutely required, with an effective concentration of 5 to 20 μM; induction was found to be possible with up to a maximum concentration of 4.4%. The calli were then maintained on a medium with a reduced 2,4-D concentration (1 μM), and were transferred every three weeks. Optimal callus induction and growth were obtained by using 3% sucrose with a combination of 0.15% Gelrite and 0.4% agar. Fronds, however, could be regenerated only on distilled water solidified with a combination of 0.4% agar and 0.15% Gelrite. On this medium, 87% of the callus expiants regenerated into fronds after four weeks of culture. These new fronds were morphologically normal but small, approximately 15 to 20% of the size of stock fronds. Continued culture of these fronds in an SH medium produced normal duckweeds, and histological examination of the cultures revealed several distinct types of callus nodules. Nonetheless, because zygotic embryogenesis inL. gibba does not produce distinct bipolar structures, the developmental pathway of frond regeneration from these nodular cultures remains unknown.     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. II. Activity and affinity of the nitrate uptake system in Pisum and Lemna in relation to nitrogen availability and nitrogen demand

Abstract Net nitrate uptake rates were measured and the kinetics calculated in non-nodulated Pisum sativum L. cv. Marma and Lemna gibba L. adapted to constant relative rates of nitrate-N additions (R_A), ranging from 0.03 to 0.27 d^?1 for Pisum and from 0.05 to 0.40 d^?1 for Lemna, V_max of net nitrate uptake (measured in the range 10 to 100 mmol m^?3 nitrate, i.e. ‘system I’) increased with R_A in the growth limiting range but decreased when R_A exceeded the relative growth rate (RGR), K_m was not significantly related to changes in R_A. On the basis of previous ¹³N-flux experiments, it is concluded that the differences in V_max at growth limiting R_A are attributable to differences in influx rates. Linear relationships between V_max and tissue nitrogen concentrations were obtained in the growth limiting range for both species, and extrapolated intercepts relate well with the previously defined minimal nitrogen concentrations for plant growth (Oscarson, Ingemarsson & Larsson, 1989). Analysis of V_max for net nitrate uptake on intact plant basis in relation to nitrogen demand during stable, nitrogen limited, growth shows an increased overcapacity at lower R_A values in both species, which is largely explained by the increased relative root size at low R_A. A balancing nitrate concentration, defined as the steady state concentration needed to sustain the relative rate of increase in plant nitrogen (R_N), predicted by R_A, was calculated for both species. In the growth limiting range, this value ranges from 3.5 mmol m^?3 (R_A 0.03 d^?1) to 44 mmol m^?3 (R_A 0.21 d^?1) for Pisum and from 0.2 mmol m^?3 (R_A 0.05 d^?1) to 5.4 mmol m^?3 (R_A 0.03 d^?1) for Lemna. It is suggested that this value can be used as a unifying measure of the affinity for nitrate, integrating the performance of the nitrate uptake system with nitrate flux and long term growth and demand for nitrogen.     

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.     

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.     

Chromium and cadmium removal from wastewater using duckweed - Lemna gibba L. and ultrastructural deformation due to metal toxicity

Phytoremediation potential of Lemna gibba was evaluated for chromium (Cr) and cadmium (Cd) under laboratory conditions for variable metal load of 1?mg/l, 3?mg/l, 5mgl, 7?mg/l and 9?mg/l, respectively, for 7 and 15?days of treatment period. Effects of both metals on structural attributes of L. gibba were also analyzed by Scanning Electron Microscopic (SEM) study. The metal removal percentage by L. gibba for Cr metal was found in the range of 37.3% to 98.6% and for cadmium it was found within the range of 81.6% to 94.6%. Bio concentration factor (BCF) of L .gibba was observed within the range of 37 to 295 for Cr metal and for Cd metal it ranged from 237 to 1144, which shows that the plant is a hyper accumulator for Cd metal and moderate accumulator for Cr metal. Statistical analysis (Two-way ANOVA) was performed on experimental results to confirm the individual effect of metal concentration and treatment period as well as cumulative effect of both factors together on percentage metal removal and on BCF. Research studies indicated that with the progress of treatment period metal removal percentage increases but increasing metal load during experiment negatively co-relates the metal removal percentage and BCF.     

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.     

Strong growth limitation of a floating plant (Lemna gibba) by the submerged macrophyte (Elodea nuttallii) under laboratory conditions

1. The asymmetric competition for light and nutrients between floating and submerged aquatic plants is thought to be key in explaining why dominance by either of these groups can be stable and difficult to change. 2. Although the shading effect of floating plants on submerged plants has been well documented, the impact of submerged plants on floating plants has been poorly explored hitherto. 3. Here, we used laboratory experiments to examine how submerged plant (Elodea nuttallii) alter nutrient conditions in the water column and how this affects the growth of floating plants (Lemna gibba). 4. We demonstrate that, at higher nutrient concentrations, Lemna is increasingly likely to outcompete Elodea. 5. Under low nutrient concentrations (0.1–2 mg N L^?1) Elodea can strongly reduce the growth of Lemna. Growth of floating plants virtually stopped in some of the experiments with Elodea. 6. Extremely reduced tissue N, Mn, chlorophyll and elongated roots indicated that the growth inhibition of Lemna by Elodea was predominantly caused by the latter’s impact on the nutrient conditions for floating plants. 7. These results strengthen the hypothesis that submerged plants can prevent colonization of a lake by floating plants.     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. I. Growth of Pisum and Lemna in relation to nitrogen

Abstract The relations between growth and internal nitrogen concentrations were investigated in nonnodulated Pisum sativum L. cv. Marma and Lemna gibba L. grown at relative rates of nitrate-N additions (R_A) varying from 0.03 to 0.27 d ¹(Pisum) and 0.05 to 0.40 d ¹ (Lemna). At R_A≤0.21 d ¹(Pisum) and ≤0.30 d ¹ (Lemna), the relative growth rate (RGR) correlated well with R_A whereas higher R_A was not met by any further increawse in growth rate. The tissue nitrogen concentrations at growth-limiting R_A increased linearly with RGR. The slope of these lines indicate a maximum nitrogen productivity (amount of biomass formed per unit nitrogen and time) of 14.4 g DW g ¹ Nd ¹ for Pisum and 15.9 g DW g ¹ N d ¹ for Lemna. Extrapolation of the plots to RGR=0 yielded intercepts of 10–15 mg N g^?1 DW for Pisum tissue, whereas for Lemna the intercepts were closer to the origin than for Pisum. These intercepts formally define a fraction of the total plant nitrogen that appears not to be active in production of new biomass, her termed ‘non-growth nitrogen’. The partitioning of nitrogen as well as biomass to the roots increased at low R_A, and is discussed in relation to activity of shoots and roots, respectively.     

Phytotoxic conjugates of indole-3-acetic acid: potential agents for biochemical selection of mutants in conjugate hydrolysis

Two strategies for selection of plant mutants altered in their ability to hydrolyze IAA conjugates are described. The rationale for both strategies is to form a conjugate between two chemical moieties in which one moiety is a toxin. The first strategy makes use of a toxic auxin analogue. For this approach a series of halogenated IAA analogues were tested for toxic properties to Lemna gibba and Zea mays. Several halogenated analogues were toxic, but only 5-Br-IAA was highly toxic to both species. The second strategy required a toxic compound with suitable functional groups to allow IAA conjugation. The mycotoxin patulin was found to be a suitable candidate. Uptake and hydrolysis of IAA-conjugates was studied in order to better define the conditions required for conjugate analogues to be effectively employed for selections.     

Photoinhibition of photosynthesis: effect of O2 and selective excitation of the photosystems in intact Lemna gibba plants

Intact Lemna gibba plants were photoinhibited under anaerobic conditions on illumination with monochromatic light which selectively excited the photosystems. Photoinhibition was less when PS 1 was excited and greatest when mainly PS 2 was excited, which suggests that PS 2 was most damaged by photoinhibition induced in complete absence of O₂ and CO₂.The illumination of plants with monochromatic light exciting PS 1, at different O₂ concentrations (in CO₂ deficient conditions), showed that PS 1 photoinhibition was increased at the low O₂ concentrations. The damage to PS 1 was more evident at 2% O₂ than at the higher O₂ concentrations.CO₂ as well as O₂ at atmospheric concentration, (air), was necessary for complete protection of the plant from photoinhibition when both photosystems were excited either separately or together.Abbreviations I irradiance, photon fluence rate - PCO photosynthetic carbon oxidation cycle - PCR photosynthetic carbon reduction cycle - PS 1 photosystem 1 - PS 2 photosystem 2