Streamlined spatial and environmental expression signatures characterize the minimalist duckweed Wolffia australiana.

Single-cell genomics permits a new resolution in the examination of molecular and cellular dynamics, allowing global, parallel assessments of cell types and cellular behaviors through development and in response to environmental circumstances, such as interaction with water and the light-dark cycle of the Earth. Here, we leverage the smallest, and possibly most structurally reduced, plant, the semiaquatic Wolffia australiana, to understand dynamics of cell expression in these contexts at the whole-plant level. We examined single-cell-resolution RNA-sequencing data and found Wolffia cells divide into four principal clusters representing the above- and below-water-situated parenchyma and epidermis. Although these tissues share transcriptomic similarity with model plants, they display distinct adaptations that Wolffia has made for the aquatic environment. Within this broad classification, discrete subspecializations are evident, with select cells showing unique transcriptomic signatures associated with developmental maturation and specialized physiologies. Assessing this simplified biological system temporally at two key time-of-day (TOD) transitions, we identify additional TOD-responsive genes previously overlooked in whole-plant transcriptomic approaches and demonstrate that the core circadian clock machinery and its downstream responses can vary in cell-specific manners, even in this simplified system. Distinctions between cell types and their responses to submergence and/or TOD are driven by expression changes of unexpectedly few genes, characterizing Wolffia as a highly streamlined organism with the majority of genes dedicated to fundamental cellular processes. Wolffia provides a unique opportunity to apply reductionist biology to elucidate signaling functions at the organismal level, for which this work provides a powerful resource.

Phytohormonal regulation determines the organization pattern of shoot aerenchyma in greater duckweed (Spirodela polyrhiza).

Airspace or aerenchyma is crucial for plant development and acclimation to stresses such as hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades are known to regulate aerenchyma formation in stems and roots under hypoxic conditions, the precise mechanisms remain unclear. Moreover, the cellular dynamics underlying airspace formation in shoots are poorly understood. We investigated the stage-dependent structural dynamics of shoot aerenchyma in greater duckweed (Spirodela polyrhiza), a fast-growing aquatic herb with well-developed aerenchyma in its floating fronds. Using X-ray micro-computed tomography and histological analysis, we showed that the spatial framework of aerenchyma is established before frond volume increases, driven by cell division and expansion. The substomatal cavity connecting aerenchyma to stomata formed via programmed cell death (PCD) and was closely associated with guard cell development. Additionally, transcriptome analysis and pharmacological studies revealed that the organization of aerenchyma in greater duckweed is determined by the interplay between PCD and proliferation. This balance is governed by spatiotemporal regulation of phytohormone signaling involving ethylene, abscisic acid, and salicylic acid. Overall, our study reveals the structural dynamics and phytohormonal regulation underlying aerenchyma development in duckweed, improving our understanding of how plants establish distinct architectural arrangements. These insights hold the potential for wide-ranging application, not only in comprehending aerenchyma formation across various plant species but also in understanding how airspaces are formed within the leaves of terrestrial plants.

Gene expression and metabolite levels converge in the thermogenic spadix of skunk cabbage.

The inflorescence (spadix) of skunk cabbage (Symplocarpus renifolius) is strongly thermogenic and can regulate its temperature at around 23 °C even when the ambient temperature drops below freezing. To elucidate the mechanisms underlying developmentally controlled thermogenesis and thermoregulation in skunk cabbage, we conducted a comprehensive transcriptome and metabolome analysis across 3 developmental stages of spadix development. Our RNA-seq analysis revealed distinct groups of expressed genes, with selenium-binding protein 1/methanethiol oxidase (SBP1/MTO) exhibiting the highest levels in thermogenic florets. Notably, the expression of alternative oxidase (AOX) was consistently high from the prethermogenic stage through the thermogenic stage in the florets. Metabolome analysis showed that alterations in nucleotide levels correspond with the developmentally controlled and tissue-specific thermogenesis of skunk cabbage, evident by a substantial increase in AMP levels in thermogenic florets. Our study also reveals that hydrogen sulfide, a product of SBP1/MTO, inhibits cytochrome c oxidase (COX)-mediated mitochondrial respiration, while AOX-mediated respiration remains relatively unaffected. Specifically, at lower temperatures, the inhibitory effect of hydrogen sulfide on COX-mediated respiration increases, promoting a shift toward the dominance of AOX-mediated respiration. Finally, despite the differential regulation of genes and metabolites throughout spadix development, we observed a convergence of gene expression and metabolite accumulation patterns during thermogenesis. This synchrony may play a key role in developmentally regulated thermogenesis. Moreover, such convergence during the thermogenic stage in the spadix may provide a solid molecular basis for thermoregulation in skunk cabbage.

Spatio-Temporal Study of Galactolipid Biosynthesis in Duckweed Using Mass Spectrometry Imaging and in vivo Isotope Labeling.

Isotope labeling coupled with mass spectrometry imaging (MSI) presents a potent strategy for elucidating the dynamics of metabolism at cellular resolution, yet its application to plant systems is scarce. It has the potential to reveal the spatio-temporal dynamics of lipid biosynthesis during plant development. In this study, we explore its application to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization-MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied after growing in 50% D2O media over a 15-day time period. When they were partially labeled after 5 d, three distinct binomial isotopologue distributions were observed corresponding to the labeling of partial structural moieties: galactose only, galactose and a fatty acyl chain and the entire molecule. The temporal change in the relative abundance of these distributions follows the expected linear pathway of galactolipid biosynthesis. Notably, their mass spectrometry images revealed the localization of each isotopologue group to the old parent frond, the intermediate tissues and the newly grown daughter fronds. Besides, two additional labeling experiments, (i) 13CO2 labeling and (ii) backward labeling of completely 50% D2O-labeled L. minor in H2O media, confirm the observations in forward labeling. Furthermore, these experiments unveiled hidden isotopologue distributions indicative of membrane lipid restructuring. This study suggests the potential of isotope labeling using MSI to provide spatio-temporal details in lipid biosynthesis in plant development.

Optimizing biochar, vermicompost, and duckweed amendments to mitigate arsenic uptake and accumulation in rice (Oryza sativa L.) cultivated on arsenic-contaminated soil.

The accumulation of arsenic (As) in rice (Oryza sativa L.) grain poses a significant health concern in Bangladesh. To address this, we investigated the efficacy of various organic amendments and phytoremediation techniques in reducing As buildup in O. sativa. We evaluated the impact of five doses of biochar (BC; BC0.1: 0.1%, BC0.28: 0.28%, BC0.55: 0.55%, BC0.82: 0.82% and BC1.0: 1.0%, w/w), vermicompost (VC; VC1.0: 1.0%, VC1.8: 1.8%, VC3.0: 3.0%, VC4.2: 4.2% and VC5.0: 5.0%, w/w), and floating duckweed (DW; DW100: 100, DW160: 160, DW250: 250, DW340: 340 and DW400: 400 g m- 2) on O. sativa cultivated in As-contaminated soil. Employing a three-factor five-level central composite design and response surface methodology (RSM), we optimized the application rates of BC-VC-DW. Our findings revealed that As contamination in the soil negatively impacted O. sativa growth. However, the addition of BC, VC, and DW significantly enhanced plant morphological parameters, SPAD value, and grain yield per pot. Notably, a combination of moderate BC-DW and high VC (BC0.55VC5DW250) increased grain yield by 44.4% compared to the control (BC0VC0DW0). As contamination increased root, straw, and grain As levels, and oxidative stress in O. sativa leaves. However, treatment BC0.82VC4.2DW340 significantly reduced grain As (G-As) by 56%, leaf hydrogen peroxide by 71%, and malondialdehyde by 50% compared to the control. Lower doses of BC-VC-DW (BC0.28VC1.8DW160) increased antioxidant enzyme activities, while moderate to high doses resulted in a decline in these activities. Bioconcentration and translocation factors below 1 indicated limited As uptake and translocation in plant tissues. Through RSM optimization, we determined that optimal doses of BC (0.76%), VC (4.62%), and DW (290.0 g m- 2) could maximize grain yield (32.96 g pot- 1, 44% higher than control) and minimize G-As content (0.189 mg kg- 1, 54% lower than control). These findings underscore effective strategies for enhancing yield and reducing As accumulation in grains from contaminated areas, thereby ensuring agricultural productivity, human health, and long-term sustainability. Overall, our study contributes to safer food production and improved public health in As-affected regions.

The evolution of the duckweed ionome mirrors losses in structural complexity.

BACKGROUND AND AIMS: The duckweeds (Lemnaceae) consist of 36 species exhibiting impressive phenotypic variation, including the progressive evolutionary loss of a fundamental plant organ, the root. Loss of roots and reduction of vascular tissues in recently derived taxa occur in concert with genome expansions of ≤14-fold. Given the paired loss of roots and reduction in structural complexity in derived taxa, we focus on the evolution of the ionome (whole-plant elemental contents) in the context of these fundamental changes in body plan. We expect that progressive vestigiality and eventual loss of roots might have both adaptive and maladaptive consequences that are hitherto unknown. METHODS: We quantified the ionomes of 34 accessions in 21 species across all duckweed genera, spanning 70 Myr in this rapidly cycling plant (doubling times are as rapid as ~24 h). We related both micro- and macroevolutionary ionome contrasts to body plan remodelling and showed nimble microevolutionary shifts in elemental accumulation and exclusion in novel accessions. KEY RESULTS: We observed a robust directional trend in calcium and magnesium levels, decreasing from the ancestral representative Spirodela genus towards the derived rootless Wolffia, with the latter also accumulating cadmium. We also identified abundant within-species variation and hyperaccumulators of specific elements, with this extensive variation at the fine (as opposed to broad) scale. CONCLUSIONS: These data underscore the impact of root loss and reveal the very fine scale of microevolutionary variation in hyperaccumulation and exclusion of a wide range of elements. Broadly, they might point to trade-offs not well recognized in ionomes.

The immediate metabolomic effects of whole-genome duplication in the greater duckweed, Spirodela polyrhiza.

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.

Responses of Vallisneria natans and Pistia stratiotes to Cu2+ and Mn2+ stress: Occurrence of caffeic acid and its degradation kinetics during chlorination.

Macrophytes are crucial in maintaining the equilibrium of aquatic ecosystems. However, the pattern of macrophyte-derived caffeic acid (CA) release under heavy metal stress is yet to be fully understood. More importantly, due to its functional groups, CA may be a precursor to the formation of disinfection by-products, posing threats to water ecology and even safety of human drinking water. This study analyzed the responses of CA released by Vallisneria natans (V. natans) and Pistia stratiotes (P. Stratiotes) when exposed to Cu2+ and Mn2+ stress. Additionally, the CA levels in two constructed wetland ponds were detected and the degradation kinetics of CA during chlorination were investigated. Results indicated that CA occurred in two constructed wetland ponds with the concentrations of 44.727 µg/L (planted with V. natans) and 61.607 µg/L (planted with P. Stratiotes). Notably, heavy metal stress could significantly affect CA release from V. natans and P. Stratiotes. In general, under Cu2+ stress, V. natans secreted far more CA than under Mn2+ stress, the level could reach up to 435.303 µg/L. However, compared to V. natans, P. Stratiotes was less affected by Cu2+ and Mn2+ stress, releasing a maximum CA content of 55.582 µg/L under 5 mg/L Mn2+ stress. Aquatic macrophytes secreted more CA in response to heavy metal stresses and protected macrophytes from harmful heavy metals. CA degradation followed the pseudo first-order kinetics model, and the chlorination of CA conformed to a second-order reaction. The reaction rate significantly accelerated as NaClO, pH, temperature and Br- concentration increased. A new pathway for CA degradation and a new DBP 2, 2, 3, 3-tetrachloropropanal were observed. These findings pointed at a new direction into the adverse effect of CA, potentially paving the way for new strategies to solve drinking water safety problems.

Uptake of perfluoroalkyl substances PFOS and PFOA by free-floating hydrophytes Pistia stratiotes L. and Eichhornia crassipes (Mart.) Solms.

The phytoremediation potential of floating aquatic plants to accumulate and remove two common PFAS from contaminated water was investigated. Free-floating hydrophytes Eichhornia crassipes and Pistia stratiotes were grown in water spiked with 0.5, 1, or 2 ppm perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS) for seven days. Both species were able to accumulate PFOA and PFOS in this time frame, with translocation factors (TF) ranging from 0.13 to 0.57 for P. stratiotes and 0.18 to 0.45 for E. stratiotes, respectively. E. crassipes accumulated a greater amount of PFOA and PFOS than P. stratiotes, with 178.9 ug PFOA and 308.5 ug PFOS removed by E. crassipes and 98.9 ug PFOA and 137.8 ug PFOS removed by P. stratiotes at the highest concentrations. Root tissue contained a higher concentration of PFOA and PFOS than shoot tissue in both species, and the concentration of PFOS was generally significantly higher than PFOA in both E. crassipes and P. stratiotes, with concentrations of 15.39 and 27.32 ppb PFOA and 17.41 and 80.62 ppb PFOS in shoots and roots of P. stratiotes and 12.59 and 37.37 ppb PFOA and 39.92 and 83.40 ppb PFOS in shoots and roots of E. crassipes, respectively. Both species may be candidates for further phytoremediation studies in aquatic ecosystems.

This study investigates the feasibility of using wetland plants for the phytoremediation of PFAS. Prior published studies examine various plant interactions with PFAS but do not evaluate remediation potential of P. stratiotes.

Changes in Metabolism and Content of Chlorophyll in Common Duckweed (Lemna minor L.) Caused by Environmental Contamination with Fluorides.

The impact of fluorine on plants remains poorly understood. We examined duckweed growth in extracts of soil contaminated with fluorine leached from chicken manure. Additionally, fluorine levels were analyzed in fresh manure, outdoor-stored manure, and soil samples at varying distances from the manure pile. Fresh manure contained 37-48 mg F- × kg-1, while soil extracts contained 2.1 to 4.9 mg F- × kg-1. We evaluated the physiological effects of fluorine on duckweed cultured on soil extracts or in 50% Murashige-Skoog (MS) medium supplemented with fluorine concentrations matching those in soil samples (2.1 to 4.9 mg F- × L-1), as well as at 0, 4, and 210 mg × L-1. Duckweed exposed to fluorine displayed similar toxicity symptoms whether in soil extracts or supplemented medium. Fluoride at concentrations of 2.1 to 4.9 mg F- × L-1 reduced the intact chlorophyll content, binding the porphyrin ring at position 32 without affecting Mg2+. This reaction resulted in chlorophyll a absorption peak shifted towards shorter wavelengths and formation of a new band of the F--chlorophyll a complex at λ = 421 nm. Moreover, plants exposed to low concentrations of fluorine exhibited increased activities of aminolevulinic acid dehydratase and chlorophyllase, whereas the activities of both enzymes sharply declined when the fluoride concentration exceeded 4.9 mg × L-1. Consequently, fluorine damages chlorophyll a, disrupts the activity of chlorophyll-metabolizing enzymes, and diminishes the plant growth rate, even when the effects of these disruptions are too subtle to be discerned by the naked human eye.

Characterization of the IAA-Producing and -Degrading Pseudomonas Strains Regulating Growth of the Common Duckweed (Lemna minor L.).

The rhizosphere represents a center of complex and dynamic interactions between plants and microbes, resulting in various positive effects on plant growth and development. However, less is known about the effects of indole-3-acetic acid (IAA) on aquatic plants. In this study, we report the characterization of four Pseudomonas strains isolated from the rhizosphere of the common duckweed (Lemna minor) with IAA-degradation and -utilization ability. Our results confirm previous reports on the negative effect of IAA on aquatic plants, contrary to the effect on terrestrial plants. P. putida A3-104/5 demonstrated particularly beneficial traits, as it exhibited not only IAA-degrading and -producing activity but also a positive effect on the doubling time of duckweeds in the presence of IAA, positive chemotaxis in the presence of IAA, increased tolerance to oxidative stress in the presence of IAA and increased biofilm formation related to IAA. Similarly, P. gessardii C31-106/3 significantly shortened the doubling time of duckweeds in the presence of IAA, while having a neutral effect in the absence of IAA. These traits are important in the context of plant-bacteria interactions and highlight the role of IAA as a common metabolite in these interactions, especially in aquatic environments where plants are facing unique challenges compared to their terrestrial counterparts. We conclude that IAA-degrading and -producing strains presented in this study might regulate IAA effects on aquatic plants and confer evolutionary benefits under adverse conditions (e.g., under oxidative stress, excess of IAA or nutrient scarcity).

Efficient Accumulation of Amylopectin and Its Molecular Mechanism in the Submerged Duckweed Mutant.

Large-scale use of fossil fuels has brought about increasingly serious problems of environmental pollution, development and utilization of renewable energy is one of the effective solutions. Duckweed has the advantages of fast growth, high starch content and no occupation of arable land, so it is a promising starchy energy plant. A new submerged duckweed mutant (sub-1) with abundant starch accumulation was obtained, whose content of amylopectin accounts for 84.04% of the starch granules. Compared with the wild type (Lemna aequinoctialis), the branching degree of starch in sub-1 mutant was significantly increased by 19.6%. Chain length DP 6-12, DP 25-36 and DP > 36 of amylopectin significantly decreased, while chain length DP 13-24 significantly increased. Average chain length of wild-type and sub-1 mutant starches were greater than DP 22. Moreover, the crystal structure and physical properties of starch have changed markedly in sub-1 mutant. For example, the starch crystallinity of sub-1 mutant was only 8.94%, while that of wild-type was 22.3%. Compared with wild type, water solubility of starch was significantly reduced by 29.42%, whereas swelling power significantly increased by 97.07% in sub-1 mutant. In order to further analyze the molecular mechanism of efficient accumulation of amylopectin in sub-1 mutant, metabolome and transcriptome were performed. The results showed that glucose accumulated in sub-1 mutant, then degradation of starch to glucose mainly depends on α-amylase. At night, the down-regulated ß-amylase gene resulted in the inhibition of starch degradation. The starch and sucrose metabolism pathways were significantly enriched. Up-regulated expression of SUS, AGPase2, AGPase3, PYG, GPI and GYS provide sufficient substrate for starch synthesis in sub-1 mutant. From the 0H to 16H light treatment, granule-bound starch synthase (GBSS1) gene was inhibited, on the contrary, the starch branching enzyme (SBE) gene was induced. Differential expression of GBSS1 and SBE may be an important reason for the decrease ratio of amylose/amylopectin in sub-1 mutant. Taken together, our results indicated that the sub-1 mutant can accumulate the amylopectin efficiently, potentially through altering the differential expression of AGPase, GBSS1, SBE, and BAM. This study also provides theoretical guidance for creating crop germplasm with high amylopectin by means of synthetic biology in the future.

Transcriptomic and Functional Analyses of Two Cadmium Hyper-Enriched Duckweed Strains Reveal Putative Cadmium Tolerance Mechanisms.

Cadmium (Cd) is one of the most toxic metals in the environment and exerts deleterious effects on plant growth and production. Duckweed has been reported as a promising candidate for Cd phytoremediation. In this study, the growth, Cd enrichment, and antioxidant enzyme activity of duckweed were investigated. We found that both high-Cd-tolerance duckweed (HCD) and low-Cd-tolerance duckweed (LCD) strains exposed to Cd were hyper-enriched with Cd. To further explore the underlying molecular mechanisms, a genome-wide transcriptome analysis was performed. The results showed that the growth rate, chlorophyll content, and antioxidant enzyme activities of duckweed were significantly affected by Cd stress and differed between the two strains. In the genome-wide transcriptome analysis, the RNA-seq library generated 544,347,670 clean reads, and 1608 and 2045 differentially expressed genes were identified between HCD and LCD, respectively. The antioxidant system was significantly expressed during ribosomal biosynthesis in HCD but not in LCD. Fatty acid metabolism and ethanol production were significantly increased in LCD. Alpha-linolenic acid metabolism likely plays an important role in Cd detoxification in duckweed. These findings contribute to the understanding of Cd tolerance mechanisms in hyperaccumulator plants and lay the foundation for future phytoremediation studies.

Do Polystyrene Nanoplastics Have Similar Effects on Duckweed (Lemna minor L.) at Environmentally Relevant and Observed-Effect Concentrations?

Although the biological effects of nanoplastics (<100 nm in size) in aquatic environments have been increasingly investigated, almost all such studies have been performed at observed-effect concentrations (higher than 1 µg/mL). The use of observed-effect concentrations of nanoplastics can provide essential data for evaluating the potential risks, but how these results apply to the effects of concentrations of nanoplastics observed in the environment remains unclear. Here, we show that exposure to both positively and negatively charged nanoplastics at the observed-effect concentration (ranging from 0 to 50 µg/mL) can result in physiological changes of Lemna minor L., a typical flowering aquatic plant species, inducing H2O2 and O2- accumulation and even cell death. However, the nanoplastics at environmentally relevant concentrations (lower than 0.1 µg/mL) had no obvious effects on phenotype of L. minor. Moreover, nanoplastics at both observed-effect and environmentally relevant concentrations were adsorbed onto the roots and fronds of the plants, whereas uptake by the roots and fronds occurred only at the observed-effect concentration. Although no phenotypic changes across 30 generations of cultivation were observed when the plants were exposed to 0.015 µg/mL nanoplastics, the expression of genes related to the response to stimuli and to oxidative and osmotic stress was upregulated under both observed-effect and environmentally relevant concentrations. Our findings suggest that the long-term presence of nanoplastics at environmentally relevant concentrations might induce some variations in the transcription level and have potential threat to floating microphytes and aquatic ecosystems.

Short-Term Test for Toxicogenomic Analysis of Ecotoxic Modes of Action in Lemna minor.

In the environmental risk assessment of substances, toxicity to aquatic plants is evaluated using, among other methods, the 7 dayLemna sp. growth inhibition test following the OECD TG 221. So far, the test is not applicable for short-term screening of toxicity, nor does it allow evaluation of toxic modes of action (MoA). The latter is also complicated by the lack of knowledge of gene functions in the test species. Using ecotoxicogenomics, we developed a time-shortened 3 day assay inLemna minor which allows discrimination of ecotoxic MoA. By examining the changes in gene expression induced by low effect concentrations of the pharmaceutical atorvastatin and the herbicide bentazon at the transcriptome and proteome levels, we were able to identify candidate biomarkers for the respective MoA. We developed a homology-based functional annotation pipeline for the reference genome ofL. minor, which allowed overrepresentation analysis of the gene ontologies affected by both test compounds. Genes affected by atorvastatin mainly influenced lipid synthesis and metabolism, whereas the bentazon-responsive genes were mainly involved in light response. Our approach is therefore less time-consuming but sensitive and allows assessment of MoA in L. minor. Using this shortened assay, investigation of expression changes of the identified candidate biomarkers may allow the development of MoA-specific screening approaches in the future.

Integrated small RNA profiling and degradome analysis of Anthurium andraeanum cultivars with different-colored spathes.

MicroRNAs (miRNAs) are known to play vital roles in coloration of leaves, flowers, and fruits in plants. However, their functions in spathe coloration are poorly known. Anthurium andraeanum is a popular ornamental plant with various spathe colors. In this study, small RNA and degradome libraries from three A. andraeanum cultivars with different-colored spathes were constructed and sequenced. Illumina sequencing resulted in 94 conserved miRNAs, and 34 novel miRNAs in total were then identified based on precursor sequences and hairpin structures. Differential expression analysis showed that 52, 51, and 49 miRNAs were differentially expressed in comparisons of orange- versus white-colored spathe, purple- versus white-colored spathe, and purple- versus orange-colored spathe, respectively. The expression patterns of miRNAs and their corresponding targets involved in spathe coloration were further analyzed, and displayed that miR156b and miR529 were highly abundant in the spathes with higher anthocyanin content. These two miRNAs co-targeted a gene encoding SPL17, which may function as a negative regulator in anthocyanin accumulation. In addition, miR408 was also abundantly expressed in purple- and orange-colored spathes, and its typical targets were also identified. This comprehensive integrated analysis provides insight into the miRNA-mediated genetic regulation in spathe coloration of A. andraeanum.

Tolerance mechanism and phytoremediation potential of Pistia stratiotes to zinc and cadmium co-contamination.

Pistia stratiotes can not only effectively remediate eutrophic water, but also displays strong absorption and bioaccumulation abilities for heavy metals. However, it has not been well-understood how the plant resists the combined stress of heavy metals. In these experiments, the morphophysiological traits, the ascorbate-glutathione (AsA-GSH) cycle, the glyoxalase system, and the contents of zinc (Zn) and cadmium (Cd) were investigated under Zn and Cd co-pollution. The AsA-GSH cycle and glyoxalase system could coordinately alleviate the oxidative and carbonyl stress, which was identified as an important tolerance mechanism. With Zn50Cd1, Zn50Cd10, Zn100Cd1, and Zn100Cd10 treatments for 18 days, 90.75-93.69% of Zn and 88.13-96.96% Cd accumulated in the roots. Treatments with Zn50Cd50, and Zn100Cd50 for 18 days resulted in a decrease of stress tolerance and chlorophyll content in leaves, an increase in plasma membrane permeability, a massive accumulation of methylglyoxal (MG), and visible toxic symptoms. Additionally, the bioaccumulation factor (BCF) for roots and shoots and the translocation factor (TF) were >1, and the content of Cd in shoots was no <100 mg·kg-1. This indicated P. stratiotes was a Cd hyperaccumulator and have great potential for the phytoremediation of heavy metal contaminated water.Novelty statement Pistia stratiotes, a cadmium hyperaccumulator, has great application potential for the phytoremediation of zinc and cadmium co-polluted water.

Differential localization of flavonoid glucosides in an aquatic plant implicates different functions under abiotic stress.

Flavonoids may mediate UV protection in plants either by screening of harmful radiation or by minimizing the resulting oxidative stress. To help distinguish between these alternatives, more precise knowledge of flavonoid distribution is needed. We used confocal laser scanning microscopy (cLSM) with the "emission fingerprinting" feature to study the cellular and subcellular distribution of flavonoid glucosides in the giant duckweed (Spirodela polyrhiza), and investigated the fitness effects of these compounds under natural UV radiation and copper sulphate addition (oxidative stress) using common garden experiments indoors and outdoors. cLSM "emission fingerprinting" allowed us to individually visualize the major dihydroxylated B-ring-substituted flavonoids, luteolin 7-O-glucoside and luteolin 8-C-glucoside, in cross-sections of the photosynthetic organs. While luteolin 8-C-glucoside accumulated mostly in the vacuoles and chloroplasts of mesophyll cells, luteolin 7-O-glucoside was predominantly found in the vacuoles of epidermal cells. In congruence with its cellular distribution, the mesophyll-associated luteolin 8-C-glucoside increased plant fitness under copper sulphate addition but not under natural UV light treatment, whereas the epidermis-associated luteolin 7-O-glucoside tended to increase fitness under both stresses across chemically diverse genotypes. Taken together, we demonstrate that individual flavonoid glucosides have distinct cellular and subcellular locations and promote duckweed fitness under different abiotic stresses.

Role of Nramp transporter genes of Spirodela polyrhiza in cadmium accumulation.

As a pollutant, Cd causes severe impact to the environment and damages living organisms. It can be uptaken from the environment by the natural resistance-associated macrophage protein (Nramp) in plants. However, the ion absorption function of Nramp transporter genes in Spirodela polyrhiza has not been reported. In this study, SpNramp1, SpNramp2, and SpNramp3 from S. polyrhiza were cloned and their functions were analyzed in S. polyrhiza and yeast. Growth parameters and physicochemical indices of wild-type and transgenic lines were measured under Cd stress. Results revealed that SpNramp1, SpNramp2, and SpNramp3 were identified as plasma membrane-localized transporters, and their roles in transporting Cd were verified in yeast. In S. polyrhiza, SpNramp1 overexpression significantly increased the content of Cd, Fe, Mn, and fresh weight. SpNramp2 overexpression increased Mn and Cd. SpNramp3 overexpression increased Fe and Mn concentrations. These results indicate that SpNramp1, SpNramp2, and SpNramp3 had a different preference for ion absorption. Two S. polyrhiza transgenic lines (OE1 and OE3) were obtained. One of them (OE1) showed a stronger accumulation ability, and the other one (OE3) exhibited tolerance capacity to Cd. This study provides new insight into the functions of SpNramp1, SpNramp2, and SpNramp3 and obtains important enrichment lines (OE1) for manipulating Cd accumulation, phytoremediation, and ecological safety.

Chemical forms governing Cd tolerance and detoxification in duckweed (Landoltia punctata).

Duckweed (Landoltia punctata) is an ideal species to restore cadmium (Cd)-polluted waters due to its fast growth and easy harvesting. To understand its tolerance and detoxification mechanism, the Cd stress responses, subcellular Cd distribution and chemically bound Cd forms (especially protein-bound Cd) were surveyed in this study. L. punctata, a potential Cd bioremediation plant, was cultured hydroponically with Cd concentrations of 0.0, 0.5, 2.0, and 5.0 mg L-1 for 5 days. The results showed that the Cd content in L. punctata increased significantly as the Cd content increased. The majority of Cd was localized in the soluble fraction (23-55%) and the cell wall fraction (21-54%), and only 14-23% of Cd was located in cell organelles. Analysis of the Cd chemical forms demonstrated that the largest portion of Cd was found in 1 M NaCl extracts, followed by d-H2O and 2% HAc extracts, indicating that Cd was mainly bound to different proteins. Albumin- and globulin-bound Cd forms were predominant, together accounting for over 80% of the total protein-bound Cd in L. punctata. These results indicate that cell wall immobilization and vacuolar dissociation of Cd are possible primary strategies for Cd biosorption and detoxification in L. punctata, which occur mainly through chemical forms changes, especially the binding of Cd to proteins.