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Summary 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. Triacylglycerol accumulation in duckweed (Lemna japonica) increased by 108‐fold to 8.7% of dry weight upon the expression of an inducible CFP‐Arabidopsis WRI1, along with constitutive expression of mouse DGAT2 and a variant sesame OLEOSIN.

Data on the new localities of Lemna minor, L. trisulca, and hybrid between L. minor and L. turionifera ( L. × japonica ) (Lemnaceae) in the north-west of the Murmansk region (north of 68.5° N), i.e., in the Pasvik State Nature Reserve and neighboring territories in the drainage basin of the Paz River, are presented. Lemna minor and L. trisulca were previously known for the region by several reports. In the north-west of the Murmansk region, two new localities of Lemna minor , one locality of L. × japonica , and ten localities of L. trisulca were found. In total, representatives of Lemna occurred in 18 studied sites: L. minor in two, L. × japonica in two, and L. trisulca in 16 ( L. minor and L. trisulca grew together in two sites). In the studied rivers and lakes, the pH range of water varied from 6.0 to 7.6, i.e., was mainly neutral (the average value was 6.9). TDS varied in the range from 17 to 251 ppm (on average, 53 ppm). The localities of L. minor in the unnamed lake near Zapolyarnyi town and in Kuetsjarvi Lake are probably the northernmost in the world, while the locality of L. trisulca in the Paz River in Klistervatn Lake is the northernmost in European Russia. In the north of the Murmansk oblast, L. minor and L. × japonica were associated only with anthropogenic eutrophicated water bodies with increased water mineralization in comparison to the background value. These taxa can be considered as indicators of high trophicity in this region. The localities of L. trisulca are mainly anthropogenic transformed water bodies. The maximum abundance of this species is also recorded in the areas of increased trophic level.

Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family’s neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela; Lemna; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.

Lemna sect. Uninerves Hegelm. consists of three species, Lemna minuta Kunth (synonym L. minuscula), L. valdiviana Phil. and L. yungensis Landolt. Lemna yungensis was discovered growing on rocks in the Yungas in Bolivia by E. Landolt and was described just 20 years ago. In the original description, Landolt reported that this species is closely related to L. valdiviana and that it is difficult to distinguish the three species on a morphological basis. Therefore, the taxonomic position and status of L. yungensis remained controversial. Here, we carried out a detailed taxonomic study, integrating approaches that include quantitative morphometry, metabolomic profiling by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) as well as molecular genetic analysis using amplified fragment length polymorphism (AFLP), and barcoding of plastidic sequences. We also investigated genome sizes of clones of the three species. Whereas L. minuta can easily be differentiated from L. valdiviana and L. yungensis, it was not possible to distinguish L. valdiviana from L. yungensis with any of the methods used. These data imply that L. yungensis is identical to L. valdiviana. Thus, the name L. yungensis should be synonymised with the name L. valdiviana, since this is the older name.

Duckweeds have been increasingly studied in recent years, both as model plants and in view of their potential applications as a new crop in a circular bioeconomy perspective. In order to select species and clones with the desired attributes, the correct identification of the species is fundamental. Molecular methods have recently provided a more solid base for taxonomy and yielded a consensus phylogenetic tree, although some points remain to be elucidated. The duckweed genus Lemna L. comprises twelve species, grouped in four sections, which include very similar sister species. The least taxonomically resolved is sect. Lemna, presenting difficulties in species delimitation using morphological and even barcoding molecular markers. Ambiguous species boundaries between Lemna minor L. and Lemna japonica Landolt have been clarified by Tubulin Based Polymorphism (TBP), with the discovery of interspecific hybrids. In the present work, we extended TBP profiling to a larger number of clones in sect. Lemna, previously classified using only morphological features, in order to test that classification, and to investigate the possible existence of other hybrids in this section. The analysis revealed several misidentifications of clones, in particular among the species L. minor, L. japonica and Lemna gibba L., and identified six putative ‘L. gibba’ clones as interspecific hybrids between L. minor and L. gibba.

Energy crises and environmental pollution have caused considerable concerns; duckweed is considered to be a promising new energy plant that may relieve such problems. Lemna aequinoctialis strain 6000, which has a fast growth rate and the ability to accumulate high levels of starch was grown in both Schenk & Hildebrandt medium (SH) and in sewage water (SW). The maximum growth rates reached 10.0 g DW m(-2) day(-1) and 4.3 g DW m(-2) day(-1), respectively, for the SH and SW cultures, while the starch content reached 39% (w/w) and 34% (w/w). The nitrogen and phosphorus removal rate reached 80% (SH) and 90% (SW) during cultivation, and heavy metal ions assimilation was observed. About 95% (w/w) of glucose was released from duckweed biomass hydrolysates, and then fermented by Angel yeast with ethanol yield of 0.19 g g(-1) (SH) and 0.17 g g(-1) (SW). The amylose/amylopectin ratios of the cultures changed as starch content increased, from 0.252 to 0.155 (SH) and from 0.252 to 0.174 (SW). Lemna aequinoctialis strain 6000 could be considered as valuable feedstock for bioethanol production and water resources purification.

The aim was to assess the ability of microcosms (laboratory-scale shallow ponds) as a post polishing stage for the remediation of artificial textile wastewater comprising two commercial dyes (basic red 46 (BR46) and reactive blue 198 (RB198)) as a mixture. The objectives were to evaluate the impact of Lemna minor L. (common duckweed) on the water quality outflows; the elimination of dye mixtures, organic matter, and nutrients; and the impact of synthetic textile wastewater comprising dye mixtures on the L. minor plant growth. Three mixtures were prepared providing a total dye concentration of 10 mg/l. Findings showed that the planted simulated ponds possess a significant (p < 0.05) potential for improving the outflow characteristics and eliminate dyes, ammonium-nitrogen (NH4-N), and nitrate-nitrogen (NO3-N) in all mixtures compared with the corresponding unplanted ponds. The removal of mixed dyes in planted ponds was mainly due to phyto-transformation and adsorption of BR46 with complete aromatic amine mineralisation. For ponds containing 2 mg/l of RB198 and 8 mg/l of BR46, removals were around 53%, which was significantly higher than those for other mixtures: 5 mg/l of RB198 and 5 mg/l of BR46 and 8 mg/l of RB198 and 2 mg/l of BR46 achieved only 41 and 26% removals, respectively. Dye mixtures stopped the growth of L. minor, and the presence of artificial wastewater reduced their development.

Both non-rooted submerged vegetation dominated by coontail (Ceratophyllum demersum) and non-rooted floating duckweed vegetation (Lemna gibba) can maintain their stable dominance in small ponds and channels. We examined the competitive interactions between them and how Ceratophyllum can sustain its stable state against floating plants in a range of nutrient concentrations. Coontail and duckweed were co-cultured in static and semi-static microcosm experiments, and their impact on the nutrients (N, P, Fe, Mn) in the water column was analysed. Coontail strongly reduced the growth of duckweed under a low nitrogen level (0.2–2 mg N L⁻¹). This reduction seems to be due to the low availability of nutrients in the water as derived from the lower nutrient concentrations in duckweed tissue or high pH in water. High nitrogen levels in semi-static media (5–10 mg N L⁻¹) resulted in an increasingly higher chance to overgrow C. demersum by L. gibba. Field observations revealed that C. demersum dominated over L. gibba in water bodies with total N below 3 mg L⁻¹, while L. gibba dominance over C. demersum occurred above 5 mg L⁻¹ total N. Ceratophyllum occurrence correlated negatively with total N in the water, while Lemna showed a positive correlation. Furthermore, the occurrence of L. gibba was negatively correlated with the frequency of C. demersum. All findings together support the theory that under a certain nutrient range, rootless submerged macrophytes have a strong potential to inhibit the dominance of floating plants in ponds, ditches and channels, and thus, they stabilize the submerged vegetation state.

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. This feature, together with high starch production and good nutritional properties, makes them suitable for several applications, including wastewater treatment, bioenergy production, or feed and food supplement. Due to their reduced morphology and great similarity between diverse species, taxonomic identification of duckweeds is a challenging issue even for experts. Among molecular genotyping methods, DNA barcoding is the most useful tool for species identification without a need for cluster analysis. The combination of two plastid barcoding loci is now considered the gold standard for duckweed classification. However, not all species can be defined with confidence by these markers, and a fast identification method able to solve doubtful cases is missing. Here we show the potential of tubulin-based polymorphism (TBP), a molecular marker based on the intron length polymorphisms of β-tubulin loci, in the genomic profiling of the genera Spirodela , Landoltia , and Lemna . Ninety-four clones were analyzed, including at least two representatives of each species of the three genera, with a special focus on the very heterogeneous species Lemna minor . We showed that a single PCR amplification with universal primers, followed by agarose gel analysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species. Cluster analysis of capillary electrophoresis–TBP data provided good separation for the remaining species, although the relationship between L. minor and Lemna japonica was not fully resolved. However, an accurate comparison of TBP profiles provided evidence for the unexpected existence of intraspecific hybrids between Lemna turionifera and L. minor , as further confirmed by amplified fragment length polymorphism and sequence analysis of a specific β-tubulin locus. Such hybrids could possibly correspond to L. japonica , as originally suggested by E. Landolt. The discovery of interspecific hybrids opens a new perspective to understand the speciation mechanisms in the family of duckweeds.

Dominant floating and submerged rootless vegetation can be regarded as alternative stable states world-wide. The competition between these two vegetation types can be strongly influenced by epiphytic algae. These algae, on the other hand, are partially controlled by grazers like snails. However, how this interaction between snails and epiphyton affects the competition between floating and submerged rootless vegetation remains rather unclear. Here, we investigate this interaction. Floating (Lemna gibba) and submerged rootless (Ceratophyllum demersum) plants were co-cultured with the presence and absence of the grazing snail Radix labiata. Biomass and nitrogen uptake of algae were strongly reduced in the presence of grazing snails. Ceratophyllum-epiphyton complex without snails reduced N and P concentration of the medium faster and had higher pH values than with the presence of grazing snails. These changes resulted in more unfavourable conditions for free-floating plants. The presence of snails indirectly increased the growth, tissue N concentration and N uptake for both Lemna and Ceratophyllum. Submerged plants together with epiphyton caused 20% more growth limitation on Lemna than Ceratophyllum alone. Structural equations modelling together with experimental results revealed that grazing snails seem to weaken the negative impact of macrophyte-epiphyton complex on Lemna. Large-scale field observations showed that the abundance of L. gibba negatively correlated with Ceratophyllum cover. Abundance of C. demersum and L. gibba negatively correlated with algal biomass; however, correlated positively with the group of larger sized grazing snails. Our findings strengthen the hypothesis that under a certain nutrient range, epiphytic algae stabilize the submerged vegetated state preventing colonization of lentic ponds by free-floating plants.