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.     

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。     

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.     

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.     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Growth rate based dose-response relationships and EC-values of ten heavy metals using the duckweed growth inhibition test (ISO 20079) with Lemna minor L. clone St

The duckweed Lemna minor L. clone St was used to investigate the effect of 10 heavy metals under the standardised test conditions of the ISO protocol 20079. By using growth rates derived from frond number (FN), fresh weight (FW), dry weight (DW), chlorophyll and carotenoid (Car) contents, concentration–response curves for all heavy metals and all growth parameters were classified. In addition, all data were fitted to obtain the inhibitions of growth rates (E_rC_x) at the level of 10%, 20% and 50% (E_rC₁₀, E_rC₂₀ and E_rC₅₀, respectively) then used to evaluate the phytotoxicity of the different heavy metals. On the basis of the E_rC₅₀ values (average ranking of all five growth parameters), the following series of phytotoxicity was detected by using molar concentrations: Ag⁺>Cd²⁺>Hg²⁺>Tl⁺>Cu²⁺>Ni²⁺>Zn²⁺>Co²⁺>Cr(VI)>As(III)>As(V).     

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.     

紫外线-B照射对小白菜生长、产量及品质的影响

在温室条件下,采用辐照度为0.4、0．5和0．6W·m^-2的紫外线-B（UV—B）对小白菜（Brassi cacampestrisssp．chinensis Makino）照射2、3和4h·d^-1,连续照射7d后,研究了UV—B照射对小白菜生长、产量及品质的影响。结果表明,停止照射后35d,各处理组小白菜的鲜质量、株高、叶片数、干物质含量、叶绿素含量、可溶性糖含量及硝酸盐含量均与对照无显著差异,但Vc含量增加,可溶性蛋白质含量降低。经UV—B照射后,下胚轴长度大于2cm的小白菜植株所占的百分率显著低于对照,说明UV—B照射可有效控制小白菜植株徒长。从子叶充分展开时用0,4—0．6W·m^-2 UV—B照射2h·d^-1,连续照射7d,既能有效控制小白菜植株徒长、提高Vc含量,又不会造成产量和品质的下降,为最佳的补充UV—B处理条件。     

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.     

Isotherm and kinetic modelling of Toluidine Blue (TB) removal from aqueous solution using Lemna minor

The aim of this study was to investigate the ability of Lemna minor for Toluidine Blue (TB) removal. Influence of the initial concentration over the removal process was considered. Experimental data have been analyzed using Langmuir, Freundlich, Dubinin–Radushkevich (D–R), and Elovich isotherm models. In addition, several kinetic models, pseudo-first-, pseudo-second-order, intraparticle and film diffusion models were considered. Langmuir and Freundlich isotherm suggested a favorable adsorption of TB by Lemna minor plants. From the D–R the mean free energy was calculated to be 11.18 kJ/mol, which indicates that TB adsorption was characterized by a chemisorption process. Kinetic studies showed that liquid film diffusion plays an important role during the process. Adsorption capacities of up to 26.69 mg/g and a high capacity of adaptation indicated that phytoremediation using Lemna minor could be a valuable alternative for dyes removal from wastewaters.     

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 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.     

Accumulated effect of elevated ultraviolet-B radiation over multiple generations of the arid-environment annual Dimorphotheca sinuata DC. (Asteraceae)

Two D. sinuata populations, established from the same seed batch, were cultured concurrently under ambient (2.5–8.9 kj m^?2 d^?1) and enhanced (4.7–11.4 kj m^?2 d^?1) ultraviolet-B radiation for two consecutive generations. The two populations, designated low- and high-UV-B cultivars, were each grown for an additional generation under both UV-B fluences at different alignments. Differences in plant performance were examined between UV-B treatments (immediate UV-B effects), cultivars (accumulated UV-B effects), alignments and their interactions. Accumulated UV-B had a greater effect on plant performance than immediate UV-B. The former induced earlier reproductive effort, substantial (up to 35%) reductions in dry mass (stems, leaves and reproductive organs), decreased stem and inflorescence production, and diminished steady-state fluorescence yields, chlorophyll a concentrations, pollen tube growth and germination of seeds set. The latter caused only diminished non-photochemical quenching, reduced chlorophyll a, soluble sugar and starch concentrations, decreased pollen germination, and increased carotenoid contents. Interactions between UV-B fluence level and alignment occurred for all measured dry mass parameters, leaf area, photosynthetic pigment and soluble sugar concentrations, and germination success of seeds set. These results indicate altered DNA integrity rather than an indirect UV-B effect of photomorphogenic origin.     

田间增加UV-B辐射对玉米光合生理的影响

自然条件下,分别增加0(CK组)、0.01(R1组)和0.015 J.m-2.s-1(R2组)3种强度的UV-B辐射处理玉米(Zea maysL.),对重庆地区C4植物玉米整个生长期内光合作用的影响进行研究。结果表明,增强UV-B辐射导致玉米幼苗叶片光合色素含量(包括叶绿素a、叶绿素b、总叶绿素和类胡萝卜素)、Hill反应活力、气孔导度、光合速率下降,且随UV-B辐射强度的增加,玉米幼苗光合作用所受抑制增强,而玉米成株所受影响不大。     

Effect of springtime solar ultraviolet-B radiation on growth of Colobanthus quitensis at Palmer Station, Antarctica

We examined the influence of solar ultraviolet‐B radiation (UV‐B; 280–315 nm) on the growth of Colobanthus quitensis plants by placing them under contrasting UV‐B filters at Palmer Station, along the Antarctic Peninsula. The filters reduced diurnal biologically effective UV‐B (UV‐B_BE) either by 83% (‘reduced UV‐B’) or by 12% (‘near‐ambient UV‐B’) over the 63 day experiment (7 November 1998–8 January 1999). Ozone column depletion averaged 17% during the experiment. Relative growth and net assimilation rates of plants exposed to near‐ambient UV‐B were 30 and 20% lower, respectively, than those of plants exposed to reduced UV‐B. The former plants produced 29% less total biomass, as a result of containing 54% less aboveground biomass. These reductions in aboveground biomass were mainly the result of a 45% reduction in shoot biomass, and a 31% reduction in reproductive biomass. Reductions in shoot biomass were owing to an 18% reduction in branch production by main shoots, while reductions in reproductive biomass were the result of a 19% reduction in individual capsule mass. Total plant leaf area was reduced by 19% under near‐ambient UV‐B, although total leaf biomass was unaffected because leaves had a greater specific leaf mass. The reduction in plant leaf area under near‐ambient UV‐B was attributable to: (1) production of 11% fewer leaves per main shoot system and plant, which resulted from an 18% reduction in branch production by main shoots. Leaf production per individual main shoot or branch was not affected; (2) shorter leaf longevity—main shoots contained 14% fewer green leaves at a given time; and (3) smaller individual leaves—leaf elongation rates were 14% slower and mature leaves were 13% shorter.     

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 exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Effect of pH,Temperature, and Lead Concentration on the Bioremoval of Lead from Water Using Lemna Minor

This study examined the ability of the aquatic plant Lemna minor (duckweed) to remove soluble lead under various laboratory conditions. In a batch process L. minor was exposed to different pH values (4.5–8.0) and temperature (15–35°C) in presence of different lead concentrations (0.1–10.0 mg L^?1) for 168 h. The amount of biomass obtained in the study period on a dry weight basis, the concentrations of lead in tissue and in medium and net uptake of lead by Lemna all have been determined in each condition. The percentages of lead uptake ratios (PMU) and bioconcentration factors (BCF) were also calculated for these conditions. Bioaccumulated lead concentrations and the PMU were obtained at lowest pH of 4.5, and at 30°C. The highest accumulated lead concentration was found at pH 4.5 as 3.599 mg Pb g^?1 in 10.0 mg L^?1. It decreased to pH 6.0, but it did not change at pH 6.0–8.0 range. The maximum lead accumulation was obtained at 30°C as 8.622 mg Pb g^?1 in 10 mg L^?1 at pH 5.0, and the minimum was at 15°C as 0.291 mg g^?1 in 0.1 mg L^?1. Lead accumulation gradually increased with increasing lead in medium, but the opposite trend was observed for PMU. Lead accumulation increased up to 50 mg L^?1, but did not change significantly in the 50.0–100.0 mg L^?1 range. The lead uptake from water was modeled and the equation fit the experimental data very well.     

不同浓度铜对紫背萍和青萍色素含量及抗氧化酶系统的影响

通过水培试验研究了不同浓度的重金属Cu对紫背萍(Spirodela polyrrhiza)和青萍(Lemna minor)的色素含量和抗氧化酶系统的影响.结果表明,低浓度Cu(0.056 mg·L^-1)的处理下,紫背萍和青萍的叶绿素a、叶绿素b和叶绿素(a+b)的含量均出现不同程度的增加,分别高出其对照11％、46％、22％和8％、15％、11％,而在高浓度Cu(0.18～5.60 mg·L^-1)的处理下,上述色素含量均显著下降,平均下降幅度分别达63％、62％、65%和46％、44％、45％.紫背萍体内丙二醛(MDA)含量为青萍的2.57倍.两种浮萍的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性均随Cu浓度的增加而呈先升后降的趋势,紫背萍体内3种酶活性在Cu浓度仅为0.18 mg·L^-1时即达峰值,随后则大幅下降,而青萍体内3种酶活性却在Cu浓度分别升高到0.56、1.0和1.0 mg·L^-1时才达到峰值.可见,在Cu胁迫下,紫背萍受Cu毒害较青萍深,且其体内抗氧化酶系统也较青萍敏感.     

He-Ne激光和增强UV-B辐射对小麦幼苗微管结合蛋白MAP65s的影响

分别采用5 mW/mm2 He-Ne激光辐照、10.08 kJ/m2·d 增强UV-B辐射及二者组合对"临优2018"小麦幼苗进行处理,通过紫外吸收法测定各处理组幼苗中微管结合蛋白MAP65s的含量,并且采用SDS-聚丙烯酰胺凝胶电泳对MAP65s蛋白进行鉴定,进一步分析He-Ne激光对增强UV-B辐射引起的小麦幼苗微管骨架损伤的修复效应.结果显示:经UV-B处理(B)后,小麦幼苗中的MAP65s蛋白含量低于对照组(CK)(P<0.01);单独激光(L)处理,MAP65s蛋白含量高于对照组(CK)(P<0.05);经He-Ne激光和UV-B复合处理(BL)后,MAP65s蛋白含量高于UV-B处理组,低于CK组.该结果表明:一定剂量的He-Ne激光辐照能够促进小麦幼苗微管结合蛋白MAP65s的合成,并且可能在一定程度上修复增强UV-B辐射对小麦幼苗微管骨架系统造成的损伤.