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Cadmium (Cd) is one of the most toxic elements to all organisms. Glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is considered an extremely important mechanism in Cd detoxification in plants. However, few studies have focused on the roles of glutamate-cysteine ligase (GSH1) and phytochelatin synthase (PCS1) in Cd accumulation and detoxification in plants. In this study, SpGSH1 and SpPCS1 were identified and cloned from Spirodela polyrhiza and analyzed their functions in yeast and S. polyrhiza via single- or dual-gene (SpGP1) overexpression. The findings of this study showed that SpGSH1, SpPCS1, and SpGP1 could dramatically rescue the growth of the yeast mutant Δycf1. In S. polyrhiza, SpGSH1 was located in the cytoplasm and could promote Mn and Ca accumulation. SpPCS1 was located in the cytoplasm and nucleus, mainly expressed in meristem regions, and promoted Cd, Fe, Mn, and Ca accumulation. SpGSH1 and SpPCS1 co-overexpression increased the Cd, Mn, and Ca contents. Based on the growth data of S. polyrhiza, it was recommended that biomass as the preferable indicator for assessing plant tolerance to Cd stress compared to frond number in duckweeds. Collectively, this study for the first time systematically elaborated the function of SpGSH1 and SpPCS1 for Cd detoxification in S. polyrhiza.

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PREMISE: In plants, whole-genome duplication (WGD) is a common mutation with profound evolutionary potential. Given the costs associated with a superfluous genome copy, polyploid establishment is enigmatic. However, in the right environment, immediate phenotypic changes following WGD can facilitate establishment. Metabolite abundances are the direct output of the cell's regulatory network and determine much of the impact of environmental and genetic change on the phenotype. While it is well known that an increase in the bulk amount of genetic material can increase cell size, the impact of gene dosage multiplication on the metabolome remains largely unknown. METHODS: We used untargeted metabolomics on four genetically distinct diploid-neoautotetraploid pairs of the greater duckweed, Spirodela polyrhiza, to investigate how WGD affects metabolite abundances per cell and per biomass. RESULTS: Autopolyploidy increased metabolite levels per cell, but the response of individual metabolites varied considerably. However, the impact on metabolite level per biomass was restricted because the increased cell size reduced the metabolite concentration per cell. Nevertheless, we detected both quantitative and qualitative effects of WGD on the metabolome. Many effects were strain-specific, but some were shared by all four strains. CONCLUSIONS: The nature and impact of metabolic changes after WGD depended strongly on the genotype. Dosage effects have the potential to alter the plant metabolome qualitatively and quantitatively, but were largely balanced out by the reduction in metabolite concentration due to an increase in cell size in this species.

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Plant-microbe remediation technique is considered as a promising technology in removal of organic pollutants and its remediation efficiency is largely affected by a variety of surrounding environmental factors. Humic acid (HA) is the complex organic substance ubiquitous in environment, which characterized by its surfactant-like micelle microstructure and various reaction activity. In our study, a plant-microbe association with high p-tert-Butylphenol (PTBP) degradation potential constructed by Spirodela polyrhiza and Sphingobium phenoxybenzoativorans Tas13 has been used, and the influence of HA on the PTBP degradation efficiency of S. polyrhiza-Tas13 association was investigated. The result showed that the presence of HA greatly improved PTBP removal efficiency of S. polyrhiza-Tas13. The reason accounted for this may be due to the presence of HA promoted bacterial cell propagation, altered bacterial cell wall permeability, increased catechol 2,3-dioxygenase (C23O) enzyme activity of strain Tas13, rather than increasing the colonization ability of strain Tas13 on to the root surface. This study will greatly facilitate the application of aquatic plant-microbe association in environmental remediation.

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Ribosomal DNA (rDNA) contains highly conserved, specifically organized sequences encoding ribosomal RNAs (rRNAs) separated by variable non-transcribed intergenic spacers (NTSs) and is abundant in eukaryotic genomes. These characteristics make the rDNA an informative molecular target to study genome organization, molecular evolution, and phylogenetics. In this study, we characterized the 5S rDNA repeats in the greater duckweed Spiroldela polyrhiza, a species known for its small size, rapid growth, highly conserved genome organization, and low mutation rate. Sequence analysis of at least 12 individually cloned PCR fragments containing the 5S rDNA units for each of six ecotypes that originated from Europe (Ukraine) and Asia (China) revealed two distinct types of 5S rDNA repeats containing NTSs of different lengths and nucleotide compositions. The shorter 5S rDNA repeat units had a highly homogeneous 400-bp NTS, with few ecotype- or region-specific single-nucleotide polymorphisms (SNPs). The longer 5S rDNA units had NTSs of 1056-1084 bp with characteristic intra- and inter-genomic variants due to specific SNPs and insertions/deletions of 4-15-bp DNA elements. We also detected significant variability in the ratio of short/long 5S rDNA variants between ecotypes of S. polyrhiza. The contrasting dynamics of the two types of 5S rDNA units, combined with the unusually low repeat copy number (for plants) in S. polyrhiza (46-220 copies per genome), shows that this species could serve as an excellent model for examining the mechanisms of concerted evolution and functional significance of rDNA variability.

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Understanding non-target-site resistance (NTSR) to herbicides represents a pressing challenge as NTSR is widespread in many weeds. Using giant duckweed (Spirodela polyrhiza) as a model, we systematically investigated genetic and molecular mechanisms of diquat resistance, which can only be achieved via NTSR. Quantifying the diquat resistance of 138 genotypes, we revealed an 8.5-fold difference in resistance levels between the most resistant and most susceptible genotypes. Further experiments suggested that diquat uptake and antioxidant-related processes jointly contributed to diquat resistance in S. polyrhiza. Using a genome-wide association approach, we identified several candidate genes, including a homolog of dienelactone hydrolase, that are associated with diquat resistance in S. polyrhiza. Together, these results provide new insights into the mechanisms and evolution of NTSR in plants.

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Duckweeds (Lemnaceae) are aquatic nongrass monocots that are the smallest and fastest-growing flowering plants in the world. While having simplified morphologies, relatively small genomes, and many other ideal traits for emerging applications in plant biotechnology, duckweeds have been largely overlooked in this era of synthetic biology. Here, we report that Greater Duckweed (Spirodela polyrhiza), when simply incubated in a solution containing plasmid-wrapped carbon nanotubes (DNA-CNTs), can directly uptake the DNA-CNTs from their growth media with high efficiency and that transgenes encoded within the plasmids are expressed by the plants─without the usual need for large doses of nanomaterials or agrobacterium to be directly infiltrated into plant tissue. This process, called the "duckweed dip", represents a streamlined, "hands-off" tool for transgene delivery to a higher plant that we expect will enhance the throughput of duckweed engineering and help to realize duckweed's potential as a powerhouse for plant synthetic biology.

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Duckweed has been highlighted as an invaluable resource because of its abilities to remove nitrogen and phosphorus from wastewater coupling with the production of high starch/protein-containing plant biomass. Duckweed recruits microbes and particularly forms a stable "core" bacterial microbiota, which greatly reduces the colonization efficiency of plant growth-promoting bacteria (PGPB). In this study, natural duckweeds were enriched in a sterilized-partially treated wastewater effluent from a poultry farm. After 24 days of cultivation, the duckweed-associated bacteria (DAB) were isolated and evaluated for their plant growth-promoting (PGP) potentials by co-cultivation with axenic Spirodela polyrhiza. Ten species were found in more than one location and could be considered candidates for the stable "core" DAB. Among them, all isolates of Acinetobacter soli, Acidovorax kalamii, Brevundimonas vesicularis, Pseudomonas toyotomiensis, and Shinella curvata increased duckweed growth in Hoagland medium. The highest PGP ability was observed in Sh. curvata W12-8 (with EPG value of 208.72%), followed by Paracoccus marcusii W7-16 (171.31%), Novosphingobium subterraneum W5-13 (156.96%), and Ac. kalamii W7-18 (156.96%). However, the highest growth promotion in the wastewater was observed when co-cultured with W7-16, which was able to increase biomass dry weight and root length of duckweed by 3.17 and 2.26 folds, respectively.

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Spirodela polyrhiza (L.) SCHLEID. has been used to treat epidemic fever, dysuria, and various skin ailments, such as measles eruptions, eczema, and pruritus, in China, Japan, and Korea. In this study, the active compounds in S. polyrhiza and their target genes were identified by network-based analysis. Moreover, the study evaluated the effects of a 70% ethanolic extract of S. polyrhiza (EESP) on skin lesions, histopathological changes, inflammatory cytokines, and chemokines in mice with contact dermatitis (CD) induced by 1-fluoro-2,4-dinitrobenzene (DNFB), and examined the inhibitory effects of EESP on mitogen-activated protein kinase (MAPK) signalling pathways. In our results, 14 active compounds and 29 CD-related target genes were identified. Among them, tumour necrosis factor (TNF) and interleukin 6 (IL-6) were identified as hub genes, and luteolin and apigenin showed a strong binding affinity with TNF (<-8 kcal/mol) and IL-6 (<-6 kcal/mol). Our in vivo studies showed that topical EESP ameliorated DNFB-induced skin lesions and histopathological abnormalities, and reduced the levels of TNF-α, interferon (IFN)-É£, IL-6, and monocyte chemotactic protein (MCP)-1 in inflamed tissues. In conclusion, our findings suggest the potential for dermatological applications of S. polyrhiza and suggest that its anti-dermatitis action is related to the inhibition of TNF and IL-6 by luteolin and luteolin glycosides.

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Aquatic environment are often contaminated with heavy metals from various industrial sources. However, physicochemical techniques for pollutant detection are limited, thus prompting the need for additional bioassays. We investigated the use of greater duckweed (Spirodela polyrhiza) as a bioindicator of metal pollution. We exposed S. polyrhiza to four pollutants (namely, silver, cadmium, copper, and chromium) and assessed metal toxicity by measuring its frond area and the length of its regrown roots. The plant displayed significant differences in both frond size and root growth in response to the four metals. Silver was the most toxic (EC50 = 23 µg L-1) while copper the least (EC50 = 365-607 µg L-1). Direct comparisons of metal sensitivity and the reliability of the two endpoint assays showed that root growth was more sensitive (lower in terms of 50% effective concentration) to chromium, cadmium, and copper, and was more reliable (lower in terms of coefficient of variation) than those for frond area. Compared to conventional Lemna-based tests, the S. polyrhiza test is easier to perform (requiring only one 24-well plate, 3 mL of medium and a 72-h exposure). Moreover, it does not require livestock cultivation/maintenance, making it more suitable for repeated measurements. Measurements of S. polyrhiza root length may be suitable for assessment when copper and chromium in municipal and industrial wastewater exceed the environmentally permissible levels.

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Material containing pectin and arabinogalactan-protein (AGP) was released and purified from Spirodela alcohol insoluble residues. Results of carbohydrate analyses and two-dimensional NMR spectroscopy suggest that this material is composed of apiogalacturonan and rhamnogalacturonan-I covalently attached to AGPs. 11B NMR spectroscopy indicated that some of the glycoses in this complex exist as their boric acid monoesters. Borate diesters were formed when the pectic-AGPs were allowed to react at pH above 6.2 with the boron-depleted pectic-AGPs, suggesting that in vitro two pectic-AGP molecules can crosslink to one another through borate. Borate diesters also formed when the pectic-AGPs were incubated with monomeric rhamnogalacturonan-II in the presence of Pb2+ ion at pH 9.2. This data presents evidence of the first wall polymer after rhamnogalacturonan-II to crosslink through borate diesters. We suggest that the formation of these borate-crosslinks may help Spirodela respond to high-pH condition.

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Progestogens and androgens have been found in many plants, but little is known about their biosynthesis and the evolution of steroidogenesis in these organisms. Here, we show that the occurrence and biosynthesis of progestogens and androgens are conserved across the viridiplantae lineage. An UHPLC-ESI-MS/MS method allowed high-throughput analysis of the occurrence and chemical conversion of progestogens and androgens in 41 species across the green plant lineage. Dehydroepiandrosterone, testosterone, and 5α-dihydrotestosterone are plants' most abundant mammalian-like steroids. Progestogens are converted into 17α-hydroxyprogesterone and 5α-pregnane-3,20-dione. Androgens are converted into testosterone and 5α-dihydrotestosterone. 17,20-Lyases, essential for converting progestogens to androgens, seem to be most effective in monocot species. Our data suggest that the occurrence of progestogens and androgens is highly conserved in plants, and their biosynthesis might favor a route using the Δ4 pathway.

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The immediate effects of plant polyploidization are well characterized and it is generally accepted that these morphological, physiological, developmental, and phenological changes contribute to polyploid establishment. Studies on the environmental dependence of the immediate effects of whole-genome duplication (WGD) are, however, scarce but suggest that these immediate effects are altered by stressful conditions. As polyploid establishment seems to be associated with environmental disturbance, the relationship between ploidy-induced phenotypical changes and environmental conditions is highly relevant. Here, we use a common garden experiment on the greater duckweed Spirodela polyrhiza to test whether the immediate effects of WGD can facilitate the establishment of tetraploid duckweed along gradients of two environmental stressors. Because successful polyploid establishment often depends on recurrent polyploidization events, we include four genetically diverse strains and assess whether these immediate effects are strain-specific. We find evidence that WGD can indeed confer a fitness advantage under stressful conditions and that the environment affects ploidy-induced changes in fitness and trait reaction norms in a strain-specific way.

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Nitrogen is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants. Here we investigated the influence of the two major sources of inorganic nitrogen, nitrate and ammonium, on the toxicity caused by excess of Mn in great duckweed, Spirodela polyrhiza. The revealed alleviating effect of ammonium on Mn-mediated toxicity, was complemented by detailed molecular, biochemical and evolutionary characterization of the species ammonium transporters (AMTs). Four genes encoding AMTs in S. polyrhiza, were classified as SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. Functional testing of the expressed proteins in yeast and Xenopus oocytes clearly demonstrated activity of SpAMT1;1 and SpAMT1;3 in transporting ammonium. Transcripts of all SpAMT genes were detected in duckweed fronds grown in cultivation medium, containing a physiological or 50-fold elevated concentration of Mn at the background of nitrogen or a mixture of nitrate and ammonium. Each gene demonstrated an individual expression pattern, revealed by RT-qPCR. Revealing the mitigating effect of ammonium uptake on manganese toxicity in aquatic duckweed S. polyrhiza, the study presents a comprehensive analysis of the transporters involved in the uptake of ammonium, shedding a new light on the interactions between the mechanisms of heavy metal toxicity and the regulation of the plant nitrogen metabolism.

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In this chapter, we present the use of Spirodela polyrhiza in experiments designed to study the evolutionary impact of whole-genome duplication (WGD). We shortly introduce this duckweed species and explain why it is a suitable model for experimental evolution. Subsequently, we discuss the most relevant steps and methods in the design of a ploidy-related duckweed experiment. These steps include strain selection, ploidy determination, different methods of making polyploid duckweeds, replication, culturing conditions, preservation, and the ways to quantify phenotypic and transcriptomic change.

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Spirodela polyrhiza (Araceae family) is a duckweed species that serves as a potential resource for feed, food, bioremediation, and pharmaceutical applications. In this study, we assessed the effects of different concentrations of melatonin (0, 0.1, 1, and 10 µM) on the growth of S. polyrhiza during in vitro culture and the metabolic profiles and productivities of useful metabolites using gas chromatography-mass spectrometry coupled with multivariable statistical analysis. We found that exogenous melatonin significantly improved the total dry weight and altered the metabolic profiles of S. polyrhiza cultures. Melatonin significantly enhanced the cellular production of useful metabolites, such as γ-aminobutyric acid, dopamine, threonine, valine, and phytosterols. The volumetric productivities (mg/L) of γ-aminobutyric acid, dopamine, campesterol, ß-sitosterol, and stigmasterol were the highest in the presence of 10 µM melatonin on day 12. Moreover, the productivities of ascorbic acid and serotonin were the highest in the presence of 1 µM melatonin on day 12. Therefore, melatonin could be used to enhance the production of biomass and useful metabolites during large-scale S. polyrhiza cultivation in cosmetic, food/feed, and pharmaceutical industries.

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With global warming, high-temperature stress has become an essential abiotic factor affecting plant growth and survival. However, little knowledge was available of the molecular mechanism that aquatic plants respond to this stress. In the present study, we explore the adaptation mechanism of Spirodela polyrhiza, a surface-water-grown duckweed species broadly distributed worldwide to high temperatures, and analyze its gene expression pattern of S. polyrhiza under heat stress. Three temperature stress treatments, including room temperature group (CK), middle high-temperature group (MTS), and high-temperature group (45 °C, HTS) were set. The results showed that the contents of SOD first increased and then decreased, and those of MDA showed an upward trend under elevated high-temperature stress. According to the transcriptome data, 3145, 3487, and 3089 differently expressed genes (DEGs) were identified between MTS and CK, HTS and CK, and HTS and MTS, respectively. The transcription factors (TFs) analysis showed that 14 deferentially expressed TFs, including HSF, ERF, WRKY, and GRAS family, were responsive to heat stress, suggesting they might play vital roles in improving resistance to heat stress. In conclusion, S. polyrhiza could resist high temperatures by increasing SOD activity and MDA at the physiological level. Several transcription factors, energy accumulation processes, and cell membranes were involved in high-temperature stress at the molecular level. Our findings are helpful in better grasping the adaptation rules of some aquatic plants to high temperatures.

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In nature, all plants live with microbes, which can directly affect their host plants' physiology and metabolism, as well as their interacting partners, such as herbivores. However, to what extent the microbiota shapes the adaptive evolution to herbivory is unclear. To address this challenge, it is essential to quantify the intra-specific variations of microbiota effects on plant fitness. Here, we quantified the fitness effects of microbiota on the growth, tolerance, and resistance to herbivory among six genotypes of the giant duckweed, Spirodela polyrhiza. We found that the plant genotypes differed in their intrinsic growth rate and tolerance, but not in their resistance to a native herbivore, the great pond snail. Inoculation with microbiota associated with S. polyrhiza growing outdoors reduced the growth rate and tolerance in all genotypes. Additionally, the microbiota treatment altered the herbivory resistance in a genotype-specific manner. Together, these data show the potential of microbiota in shaping the adaptive evolution of plants.

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The predominantly vegetative propagating duckweeds are of growing commercial interest. Since clonal accessions within a respective species can vary considerably with respect to their physiological as well as biochemical traits, it is critical to be able to track the clones of species of interest after their characterization. Here, we compared the efficacy of five different genotyping methods for Spirodela polyrhiza, a species with very low intraspecific sequence variations, including polymorphic NB-ARC-related loci, tubulin-gene-based polymorphism (TBP), simple sequence repeat variations (SSR), multiplexed ISSR genotyping by sequencing (MIG-seq), and low-coverage, reduced-representation genome sequencing (GBS). Four of the five approaches could distinguish 20 to 22 genotypes out of the 23 investigated clones, while TBP resolved just seven genotypes. The choice for a particular method for intraspecific genotyping can depend on the research question and the project budget, while the combination of orthogonal methods may increase the confidence and resolution for the results obtained.

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Growth-regulating factors (GRFs) are plant-specific transcription factors that play essential roles in regulating plant growth and stress response. The GRF gene families have been described in several terrestrial plants, but a comprehensive analysis of these genes in diverse aquatic species has not been reported yet. In this study, we identified 130 GRF genes in 13 aquatic plants, including floating plants (Azolla filiculoides, Wolffia australiana, Lemna minuta, Spirodela intermedia, and Spirodela polyrhiza), floating-leaved plants (Nymphaea colorata and Euryale ferox), submersed plants (Zostera marina, Ceratophyllum demersum, Aldrovanda vesiculosa, and Utricularia gibba), an emergent plant (Nelumbo nucifera), and an amphibious plant (Cladopus chinensis). The gene structures, motifs, and cis-acting regulatory elements of these genes were analyzed. Phylogenetic analysis divided these GRFs into five clusters, and ABRE cis-elements were highly enriched in the promoter region of the GRFs in floating plants. We found that abscisic acid (ABA) is efficient at inducing the turion of Spirodela polyrhiza (giant duckweed), accompanied by the fluctuated expression of SpGRF genes in their fronds. Our results provide information about the GRF gene family in aquatic species and lay the foundation for future studies on the functions of these genes.

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Duckweed is a kind of aquatic plant with the characteristics of high nutritional value and medicinal benefits. However, most researches focused on the natural germplasms. The underlying metabolic pathway remains to be systematically elaborated in duckweed. In our laboratory, one reddish-purple mutant with high-flavonoids was screened from a mutant library of Spirodela polyrhiza 6068, named 68-red. The content of anthocyanins and proanthocyanidins in 68-red mutant increased by 563.47% and 231.19%, respectively, compared to wild type. It is interesting that cynaroside and orientin content were significantly increased, in contrast, apigetrin and vitexin were decreased in 68-red mutant. Considering this, metabolome and transcriptome were employed to explore the flavonoids biosynthetic pathway. Here, a total of 734 metabolites were identified in the wild type and 68-red mutant. Among which, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside were significantly accumulated, which were positively correlated with deep reddish-purple of 68-red mutant. In addition, proanthocyanidins (B1, B2, B3, B4, C1, C2), flavonoid and its glycosides (11 luteolin and its glycosides, 14 quercetin and its glycosides, 14 kaempferol and its glycosides, 2 apigenin glycosides) were significantly accumulated, 2 apigenin glycosides were down-regulated in 68-red mutant. The transcriptome data and qRT-PCR indicated that 16 enzyme genes in flavonoids biosynthetic pathway (PAL, C4H, CHSs, F3H, ANS, ANR, F3'Hs, DFRs, LAR, GT1, BZ1) were significantly up-regulated in 68-red mutant. Correlation analysis found that three copies of F3'H gene play important roles in the synthesis of anthocyanins, luteolin and apigenin glycosides. In conclusion, the 68-red mutant is a high quality germplasm resources for food and medical industry. Metabolome and transcriptome provide new insight for exploring the enzyme genes and functional metabolites in duckweed.

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