Science

Duckweeds are a common macrophyte in paddy and aquatic environments. Here, we investigated arsenic (As) accumulation, speciation and tolerance of the rootless duckweed Wolffia globosa and its potential for As phytofiltration. When grown with 1 microm arsenate, W. globosa accumulated two to 10 times more As than four other duckweed or Azolla species tested. W. globosa was able to accumulate > 1000 mg As kg(-1) in frond dry weight (DW), and tolerate up to 400 mg As kg(-1) DW. At the low concentration range, uptake rate was similar for arsenate and arsenite, but at the high concentration range, arsenite was taken up at a faster rate. Arsenite was the predominant As species (c. 90% of the total extractable As) in both arsenate- and arsenite-exposed duckweed. W. globosa was more resistant to external arsenate than arsenite, but showed a similar degree of tolerance internally. W. globosa decreased arsenate in solution rapidly, but also effluxed arsenite. Wolffia globosa is a strong As accumulator and an interesting model plant to study As uptake and metabolism because of the lack of a root-to-frond translocation barrier.

ABSTRACT The phytoaccumulation and phytotoxicity of heavy metals, cadmiun (Cd), and chromium (Cr) on a common duckweed, Wolffia globosa, were studied. W. globosa were cultured in 3% Hoagland's nutrient medium, which was supplemented with 1, 2, 4, and 8 mg/L of Cd and Cr and were separately harvested after 3, 6, 9, and 12 days. The accumulation of Cd and Cr in W. globosa showed significant increases when the exposure time and metal concentration were increased. The effects of Cd and Cr on the biomass productivity and total chlorophyll content in W. globosa indicated that there were significant decreases in the biomass productivity and total chlorophyll content when the exposure time and metal concentration were increased.

Species of the genus Wolffia are traditionally used as human food in some of the Asian countries. Therefore, all 11 species of this genus, identified by molecular barcoding, were investigated for ingredients relevant to human nutrition. The total protein content varied between 20 and 30% of the freeze-dry weight, the starch content between 10 and 20%, the fat content between 1 and 5%, and the fiber content was ~25%. The essential amino acid content was higher or close to the requirements of preschool-aged children according to standards of the World Health Organization. The fat content was low, but the fraction of polyunsaturated fatty acids was above 60% of total fat and the content of n-3 polyunsaturated fatty acids was higher than that of n-6 polyunsaturated fatty acids in most species. The content of macro- and microelements (minerals) not only depended on the cultivation conditions but also on the genetic background of the species. This holds true also for the content of tocopherols, several carotenoids and phytosterols in different species and even intraspecific, clonal differences were detected in Wolffia globosa and Wolffia arrhiza. Thus, the selection of suitable clones for further applications is important. Due to the very fast growth and the highest yield in most of the nutrients, Wolffia microscopica has a high potential for practical applications in human nutrition.

Summary 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 boundary-layer model has been used to describe the movement of dissolved oxygen through a vegetation mat of Wolffia sp. or Lemna minor. In an environment where light and temperature are held constant, the reaeration rate of the water below the mat is a linear function of mat thickness. In the light, the rate of oxygen transfer into water through a 10.5-mm mat is 4.4 times higher for Wolffia and 2.9 times higher for L. minor than in the dark. In the light the rate of oxygen transfer through Wolffia mats is significantly lower in August and September than in early summer. A seasonal shift in transport rates through L. minor mats has not been conclusively established. The efficiency of gas transfer through washed Wolffia and L. minor mats 8 to 16 mm in thickness varies from 4.0 to 47.0% of the estimated gas exchange rate for a small protected lake. The rate of gas transfer through the mat is higher than might be expected considering the close packing of the component fronds. The shape of the oxygen profile...

Cadmium (Cd) pollution around the world is a serious issue demanding acceptable solutions, one of which is phytoremediation that is both cost-effective and eco-friendly. Removal of Cd from contaminated water using plants with high growth rates and sufficient Cd accumulation abilities could be an appropriate choice. Here, we investigated a potential Cd accumulator, Wolffia, a rootless duckweed with high growth rate. Cd uptake, accumulation, tolerance, and phytofiltration ability by Wolffia globosa were examined. Furthermore, the effects of arsenic (As) on Cd uptake and phytofiltration by W. globosa were also studied. Cd uptake kinetics showed a linear pattern and a hyperbolic pattern without a plateau in lower (0–2 μM) and higher (0–200 μM) Cd concentration ranges, respectively, suggesting rapid Cd uptake by W. globosa. Cd accumulation ability by W. globosa was higher at Cd concentrations < 10 μM than at >10 μM. All the five species of Wolffia exposed to 1 μM Cd for 5 days accumulated > 500 mg Cd kg−1 DW. Ten gram fresh W. globosa could diminish almost all the Cd (2 μM) in a 200 mL solution. This enormous accumulation ability was mostly due to passive adsorption of Cd by the apoplast. Arsenic had no significant effect on Cd uptake and phytofiltration. The fresh fronds also showed a great As extracting ability. The results indicated that Wolffia is a strong Cd accumulator and has great Cd phytoremediation potential. Therefore, this plant can be used in fresh aquatic environments co-contaminated by low-levels of Cd and As. Supplemental materials are available for this article. Go to the publisher's online edition of International Journal of Phytoremediation to view the supplemental file.

Nitrate reductase was not found to be present in or associated with partially purified, intact chloroplasts aqueously isolated from Wolffia arrhiza. Such chloroplasts are capable of using nitrite but not nitrate as an electron acceptor during light-stimulated electron transport in the absence of additional cytoplasmic components. When nitrite acts as an electron acceptor under these conditions, on the average 1.5 moles of oxygen are evolved per mole of nitrite reduced by the chloroplasts, indicating a probable reduction of nitrite to ammonia. Chloroplasts ruptured by osmotic shock fail to reduce nitrite in the absence of additional components.

Flowering in Wolffia microscopica, a short-day plant, could be induced with salicylic acid (SA), under long days. Aspirin, benzoic acid and salicylaldoxime were also effective for induction of flowering in this duckweed. Amonsgt these, SA is the most effective compound, as it could induce flowering even at 10 -7 M. Flowering was further enhanced when Wolffia fronds were subjected to short days, in the presence of SA. However, SA neither showed any effect on flowering ofW. microscopica in the absence of EDTA in the nutrient medium, nor could it, by itself, support even the vegetative growth. The probable mechanism of action of SA has also been discussed. It appears that the effect cannot be due simply to chelation of metal ions and perhaps the salicyl moiety itself exerts a specific effect.

In Wolffia arrhiza {Lemnaceae) growing on municipal tap water (rich in mineral but poor in organic components) and thus being exclusively photoautotrophic, IAA (3-indolilacetic acid) and SA (salicylic acid) strongly stimulated action on the content of chlorophylls a and b and carotenoids (especially β-carotene and lutein + zeaxanthin). On the other hand, the chemical analogues of IAA, i.e. PAA (phenylacetic acid) and NAA (α-naphtylacetic acid), had a generally inhibitory effect on chlorophyll and carotenoid contents. In Wolffia arrhiza growing on raw waste water and a suspension of activated sludge from a sewage treatment plant (rich in organic substances) characterized by mixotrophism (that is photo- and heterotrophism) PAA had the highest stimulative action on the chlorophyll a and b content, SA negligible, whereas NAA had an inhibitory effect. IAA had a slight stimulative effect on raw sewage but inhibitory on activated sludge. Also, the greatest stimulative effect on carotenoids content was exerted by PAA; SA had a slight stimulative effect while IAA and, to a greater extent NAA had a clearly inhibitory influence.

ABSTRACT The biosorption of cadmium (Cd) and chromium (Cr) by using dried Wolffia globosa biomass were investigated using batch technique. The effects of concentration and pH solution on the adsorption isotherm were measured by determining the adsorption isotherm at initial metal concentrations from 10 to 400 mg/L and pH 4 to 7 for Cd, and pH 1.5 to 6 for Cr. The adsorption equilibria were found to follow Langmuir models. The maximum adsorption capacity (Xm) at pH 7 in W. globosa - Cd system was estimated to be 80.7 mg/g, while the maximum removal achieved at pH 4, pH 5, and pH 6 were 35.1, 48.8, and 65.4 mg/g, respectively. The Xm at pH 1.5 in W. globosa - Cr system was estimated to be 73.5 mg/g, while the maximum removal achieved at pH 3, pH 5, and pH 6 were 47.4, 33.1, and 12.9 mg/g, respectively. The effects of contact times on Cd and Cr sorption indicated that they were absorbed rapidly and more efficiently at lower concentrations.

Speciation is a key determinant in the toxicity, behavior, and fate of arsenic (As) in the environment. However, little is known about the transformation of As species mediated by floating macrophytes and the phyllosphere bacteria in aquatic and wetland environment. In this study, Wolffia australiana, a rootless floating duckweed, was cultured with (W+B) or without (W-B) phyllosphere bacteria to investigate its ability in arsenite (As(III)) oxidation. Results showed that sterile W. australiana did not oxidize As(III) in the growth medium or in plant tissue, whereas W. australiana with phyllpsphere bacteria displayed substantial As(III) oxidation in the medium. Quantitative PCR of As redox-related functional genes revealed the dominance of the arsenite oxidase (aioA) gene in the phyllosphere bacterial community. These results demonstrate that the phyllosphere bacteria were responsible for the As(III) oxidation in the W+B system. The rapid oxidation of As(III) by the phyllosphere bacterial community may suppress As accumulation in plant tissues under phosphate rich conditions. The aioA gene library showed that the majority of the phyllosphere arsenite-oxidizing bacteria related either closely to unidentified bacteria found in paddy environments or distantly to known arsenite-oxidizing bacteria. Our research suggests a previously overlooked diversity of arsenite-oxidizing bacteria in the phyllosphere of aquatic macrophytes which may have a substantial impact on As biogeochemistry in water environments, warranting further exploration.

Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth.

Light and electron microscope studies of Wolffia columbiana Karsten and Wolffia borealis (Engelmann ex Hegelmaier) Landolt & O. Wildi vegetative stages were undertaken. Asexual reproduction in both species involves the budding of daughter fronds from a meristematic region in the reproductive pouch of a mother frond. A single Wolffia plant (mother plus daughter plus granddaughter fronds) is composed of six to seven individuals at various developmental stages. The mesophyll of W. columbiana (especially in older fronds) is composed of thin-walled cells with few organelles; chloroplasts are concentrated instead in the epidermis and have a normal ultrastructure consisting of both appressed and unappressed thylakoids. Anticlinal cell walls of adjacent W. columbiana epidermal cells have elaborate cell wall ingrowths characteristic of transfer cells. Wolffia borealis mesophyll cells vary in size from small cells toward the dorsum to large cells toward the ventrum. Chloroplasts are concentrated in the small dorsal mesophyll cells and lack significant thylakoid appression. Transfer cells are absent in W. borealis. X-ray microanalysis indicated high levels of calcium on the W. borealis surface, with even higher concentrations in the punctae cells. Calcium oxalate was not detected. The W. columbiana transfer cells and the agranal W. borealis chloroplasts are features not previously reported in the Lemnaceae.

The mechanism of the light-stimulated absorption of nitrate by Wolffia arrhiza was studied. The nitrate-absorption mechanism in ammonium-grown plants is stimulated by the presence of nitrate. In a manner similar to the absorption of many other ions, the absorption of nitrate follows a typical biphasic pattern in relation to external nitrate concentration. Mechanism 1 is effective at nitrate concentrations up to 0.5 to 0.75 mM and mechanism 2 becomes operative at higher nitrate levels. Light stimulates the absorption of nitrate independently of the effect of light on the reduction of nitrate. The effects of uncouplers, inhibitors, and light of wavelengths of 700 nm or longer indicate that nitrate absorption by Wolffia cells is reduced when non-cyclic electron transport is blocked. It is postulated that under this condition, ATP in the chloroplast (produced by cyclic photophosphorylation) may be less readily transported across the chloroplast envelope than when non-cyclic electron transport is proceeding.

Background: Rare plants that contain corrinoid compounds mostly comprise cobalamin analogues, which may compete with cobalamin (vitamin B12 (B12)) metabolism. We examined the presence of B12 in a cultivated strain of an aquatic plant: Wolffia globosa (Mankai), and predicted functional pathways using gut-bioreactor, and the effects of long-term Mankai consumption as a partial meat substitute, on serum B12 concentrations. Methods: We used microbiological assay, liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic bioreactors for the B12 experiments. We explored the effect of a green Mediterranean/low-meat diet, containing 100 g of frozen Mankai shake/day, on serum B12 levels during the 18-month DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean diet groups. Results: The B12 content of Mankai was consistent at different seasons (p = 0.76). Several cobalamin congeners (Hydroxocobalamin(OH-B12); 5-deoxyadenosylcobalamin(Ado-B12); methylcobalamin(Me-B12); cyanocobalamin(CN-B12)) were identified in Mankai extracts, whereas no pseudo B12 was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with a genome containing a KEGG ortholog involved in microbial B12 metabolism were observed, compared with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n = 294 participants; retention-rate = 89%; baseline B12 = 420.5 ± 187.8 pg/mL), serum B12 increased by 5.2% in control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets (p = 0.025 between extreme groups). Conclusions: Mankai plant contains bioactive B12 compounds and could serve as a B12 plant-based food source.

Since duckweed (Lemnaceae family) is a valuable target plant for various applications including waste water treatment and food purposes, the expression of homologous or heterologous proteins may offer an extended range of application. Therefore, the feasability of transformation of Wolffia columbiana (Lemnaceae) by Agrobacterium tumefaciens-mediated gene transfer has been elucidated. Several methods were tested to increase the accessibility of the plant cells for the infecting Agrobacterium tumefaciens strain LBA4404, harboring p35SGUSINT: corundum- and gold particle-treatment, vacuum infiltration and disintegration of the fronds. The resulting overall transformation efficieney was higher than without any treatment, reaching an average of 3.9 % of all fronds showing GUS staining. Induction of Agrobacterium's vir genes by media conditions as well as the presence of 0.6 M mannitol during infection resulted in a clear increase of transformation efficiency. Max. approx. 30 %, average 15-20 % of fronds showing GUS staining were obtained both with corandum-treated as well as with vacuum infiltrated fronds, but transformation pattern was different. Whereas in the former mainly epidermal and subepidermal cells were transformed, the latter showed, in addition, transformed inner frond cells, including the meristematic region. Disintegration of the fronds, followed by vacuum infiltration, led to whole GUS-stained areas of the frond fragments. The results as such and the observed transformation patterns will serve as a basis for offering good conditions either in the in vivo - or the in-vitro-regeneration of transgenic duckweed fronds.

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 μg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

The purpose of this study was twofold: our London study focused on the life history of W. australiana looking specifically at the length of life and ability of each plant to produce new fronds. Subsequent to our life history study, plants at Ithaca College flowered and we began observations on anatomy and flowering. This work will serve as background for a study of aging and affects of heavy metals on Wolffia australiana

OBJECTIVE To compare the postprandial and overnight glycemic response using a novel green aquatic plant thought to provide a dietary source for high-quality protein, with an iso-carbohydrate/protein/caloric dairy shake. RESEARCH DESIGN AND METHODS This is a randomized controlled crossover trial among 20 abdominally obese participants (age 51.4 years; fasting plasma glucose 110.9 mg/dL), who were allocated to replace dinner with either, first, a green shake containing Wolffia globosa duckweed (Mankai: specific-strain) or an iso-carbohydrate/protein/calorie yogurt shake. A 2-week flash glucose-monitoring system was used to assess postmeal glucose dynamics (6 net administration days; 97 observation days in total). We further obtained from each participant dietary/daily activity/satiety scale/sleep logs. Participants were recruited from the green-Mediterranean diet arm of the 18-month Dietary Intervention Randomized Controlled Trial-Polyphenols Unprocessed (DIRECT-PLUS) study. RESULTS Wolffia globosa Mankai elicited a lower postprandial glucose peak compared with yogurt (∆peak = 13.4 ± 9.2 vs. 19.3 ± 15.1 mg/dL; P = 0.044), which occurred later (77.5 ± 29.2 vs. 59.2 ± 28.4 min; P = 0.037) and returned faster to baseline glucose levels (135.8 ± 53.1 vs. 197.5 ± 70.2 min; P = 0.012). The mean post–net incremental area under the curve (netAUC) was lower with Wolffia globosa up to 60 and 180 min (netAUC 60 min: 185.1 ± 340.1 vs. 441.4 ± 336.5 mg/dL/min, P = 0.005; netAUC  180 min: 707.9 ± 1,428.5 vs. 1,576.6 ± 1,810.1 mg/dL/min, P = 0.037). A Wolffia globosa–based shake replacing dinner resulted in lower next-morning fasting glucose levels (83.2 ± 0.8 vs. 86.6 ± 13 mg/dL; P = 0.041). Overall, postprandial glucose levels from the shake administration until the next morning were lower in the Wolffia globosa Mankai green shake compared with the yogurt shake (P < 0.001). Overnight sleep duration was similar (378.2 ± 22.4 vs. 375.9 ± 28.4 min; P = 0.72), and satiety rank was slightly higher for the Wolffia globosa shake compared with the yogurt shake (7.5 vs. 6.5; P = 0.035). CONCLUSIONS Wolffia globosa Mankai duckweed may serve as an emerging alternative plant protein source with potential beneficial postprandial glycemic effects.

The present Present experiment was conducted for 75 days in triplicates groups in 18 aquaria of 50 L each to study the effect of Wolffia arrhiza and Spirulina platensis on growth and pigmentation of Botia dario. Six isonitrogenous diets were prepared with 350 g/kg crude protein (CP) level. Diet 1 (T1) was prepared without fortification of spirulina and wolffia. T2 diet was prepared with spirulina as supplement of carotenoids. Similarly, T3, T4, T5 and T6 diets were prepared by substituting 25, 50, 75 and 100 g/kg of CP from spirulina with wolffia, respectively. Significant differences (p   .05). Thus, it can be concluded that diet containing 100 g/kg spirulina can be effective for better growth while diet containing 25 g/kg spirulina and 150 g/kg wolffia can be effective for higher survival and pigmentation in Botia dario.

For the first time to our knowledge, we demonstrate that whole angiosperm individuals can survive gut passage through birds, and that this occurs in the field. Floating plants of the genus Wolffia are the smallest of all flowering plants. Fresh droppings of white-faced whistling duck Dendrocygna viduata (n = 49) and coscoroba swan Coscoroba coscoroba (n = 22) were collected from Brazilian wetlands. Intact Wolffia columbiana were recovered from 16% of D. viduata and 32% of Coscoroba samples (total = 164 plantlets). The viability of plants was tested, and asexual reproduction was confirmed. Wolffia columbiana is an expanding alien in Europe. Avian endozoochory of asexual angiosperm propagules may be an important, overlooked dispersal means for aquatic plants, and may contribute to the invasive character of alien species.