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Ranunculus trichophyllus is an amphibious plant that produces thin and cylindrical leaves if grown under water but thick and broad leaves if grown on land. We found that such heterophylly is widely controlled by two plant hormones, abscisic acid (ABA) and ethylene, which control terrestrial and aquatic leaf development respectively. Aquatic leaves produced higher levels of ethylene but lower levels of ABA than terrestrial leaves. In aquatic leaves, their distinct traits with narrow shape, lack of stomata, and reduced vessel development were caused by EIN3-mediated overactivation of abaxial genes, RtKANADIs, and accompanying with reductions of STOMAGEN and VASCULAR-RELATED NAC-DOMAIN7 (VDN7). In contrast, in terrestrial leaves, ABI3-mediated activation of the adaxial genes, RtHD-ZIPIIIs, and STOMAGEN and VDN7 established leaf polarity, and stomata and vessel developments. Heterophylly of R.trichophyllus could be also induced by external cues such as cold and hypoxia, which is accompanied with the changes in the expression of leaf polarity genes similar to aquatic response. A closely-related land plant R. sceleratus did not show such heterophyllic responses, suggesting that the changes in the ABA/ethylene signaling and leaf polarity are one of key evolutionary steps for aquatic adaptation.

Ranunculus sceleratus L.(RS) has shown various pharmacological effects in traditional Chinese medicine. In our previous study, the positive therapeutic effect on α-naphthylisothiocyanate induced intrahepatic cholestasis in rats was obtained using TianJiu treatment with fresh RS. However, the chemical profile of RS has not been clearly clarified, which impedes the research progress on the therapeutic effect of RS. Herein, an ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed to rapidly separate and identify multiple constituents in the 80% methanol extract of RS. A total of sixty-nine compounds (19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) were successfully characterized. A total of 12 of these compounds were unambiguously identified by standard samples. Their mass spectrometric fragmentation pathways were investigated. It is worth noting that flavonoids and lignans were identified for the first time in RS. In this study, we successfully provide the first comprehensive report on identifying major chemical constituents in RS by UHPLC-Q-Orbitrap HRMS. The obtained results enrich the RS chemical profile, paving the way for further phytochemical study, quality control, and pharmacological investigation of RS.

In this study, the seed germination and dormancy characteristics of three Ranunculus species native to Korea ( R. cantoniensis , R. chinensis , and R. sceleratus ) were determined to establish an effective dormancy-breaking protocol. The results revealed that the R. cantoniensis , R. chinensis , and R. sceleratus seeds responded differently to treatment with gibberellins (GAs) and stratification. Warm stratification at 20℃ increased the germination percentage and partially broke the dormancy of the R. cantoniensis seeds; moreover, the germination temperature range of the seeds expanded upon treatment with high-concentration GA 4 + 7 . Meanwhile, neither cold nor warm stratification was effective at breaking the dormancy of R. chinensis seeds; however, high-concentration GA 4 + 7 treatment broke the dormancy of the seeds by expanding the germination temperature range. Lastly, the R. sceleratus seeds did not respond to the treatment with GAs; however, their germination percentage increased after undergoing warm stratification at 25℃. The seeds of these three species were characterized as exhibiting nondeep morphophysiological dormancy. The findings of this study enhance our understanding of the temperature-related germination responses of the three Ranunculus species, offering valuable insights into their conservation and utilization.

The novel methods for efficient plant regeneration via direct somatic embryogenesis (SE) and SE-mediated transformation system under high concentration of NAA in Ranunculus sceleratus were established. On MS media containing a high concentration of NAA (10.0 mg/L) in the dark, all inoculated explants (root, stem and leaf) formed somatic embryos at high frequencies, respectively, 66.03, 126.47 and 213.63 embryoids per explant, and 100% of the embryoids developed into plantlets on 1/2 MS rooting media. Morphological and histological analyses revealed that SE in R. sceleratus followed a classical pattern. All inoculated explants can be used as receptors for genetic transformation in R. sceleratus , through direct SE-mediated method after Agrobacterium infection. RcLEC1-B , as a marker gene, changed the number and morphology of flower organs and the development of cuticle in R. sceleratus , which indicated that the efficient transgenic system of R. sceleratus was established. To our knowledge, this is the first observation that both direct SE and transgenic transformation system, via induction of a single plant growth regulator, have been successfully constructed in R. sceleratus .

Ranunculus sceleratus (family: Ranunculaceae) is a medicinally and economically important plant; however, gaps in taxonomic and species identification limit its practical applicability. This study aimed to sequence the chloroplast genome of R. sceleratus from Republic of Korea. Chloroplast sequences were compared and analyzed among Ranunculus species. The chloroplast genome was assembled from Illumina HiSeq 2500 sequencing raw data. The genome was 156,329 bp and had a typical quadripartite structure comprising a small single-copy region, a large single-copy region, and two inverted repeats. Fifty-three simple sequence repeats were identified in the four quadrant structural regions. The region between the ndhC and trnV-UAC genes could be useful as a genetic marker to distinguish between R. sceleratus populations from Republic of Korea and China. The Ranunculus species formed a single lineage. To differentiate between Ranunculus species, we identified 16 hotspot regions and confirmed their potential using specific barcodes based on phylogenetic tree and BLAST-based analyses. The ndhE, ndhF, rpl23, atpF, rps4, and rpoA genes had a high posterior probability of codon sites in positive selection, while the amino acid site varied between Ranunculus species and other genera. Comparison of the Ranunculus genomes provides useful information regarding species identification and evolution that could guide future phylogenetic analyses.

The aim of the present study was to evaluate tolerance to salinity and different heavy metals as well as the phytoextraction potential of Ranunculus sceleratus plants from a brackish coastal sandy beach habitat. Four separate experiments were performed with R. sceleratus plants in controlled conditions: (1) the effect of NaCl gradient on growth and ion accumulation, (2) the effect of different Na+ and K+ salts on growth and ion accumulation, (3) heavy metal tolerance and metal accumulation potential, (4) the effect of different forms of Pb salts (nitrate and acetate) on plant growth and Pb accumulation. A negative effect of NaCl on plant biomass was evident at 0.5 g L−1 Na+ and growth was inhibited by 44% at 10 g L−1 Na+, and this was associated with changes in biomass allocation. The maximum Na+ accumulation (90.8 g kg−1) was found in the stems of plants treated with 10 g kg−1 Na+. The type of anion determined the salinity tolerance of R. sceleratus plants, as Na+ and K+ salts with an identical anion component had a comparable effect on plant growth: nitrates strongly stimulated plant growth, and chloride treatment resulted in slight but significant growth reduction, but plants treated with nitrites and carbonates died within 4 and 5 weeks after the full treatment, respectively. The shoot growth of R. sceleratus plants was relatively insensitive to treatment with Mn, Cd and Zn in the form of sulphate salts, but Pb nitrate increased it. Hyperaccumulation threshold concentration values in the leaves of R. sceleratus were reached for Cd, Pb and Zn. R. sceleratus can be characterized as a shoot accumulator of heavy metals and a hyperaccumulator of Na+. A relatively short life cycle together with a high biomass accumulation rate makes R. sceleratus useful for dynamic constructed wetland systems aiming for the purification of concentrated wastewaters.

The current study addressed the heavy metals accumulation potentials of seven perennial aquatic macrophytes (Cyperus alopecuroides, Echinochloa stagnina, Eichhornia crassipes, Ludwigia stolonifera, Phragmites australis, Ranunculus sceleratus and Typha domingensis) and the pollution status of three drains (Amar, El-Westany and Omar-Beck) in the Nile Delta of Egypt. Nine sites at each drain were sampled for sediment and plant analyses. Concentrations of eight metals (Fe, Cu, Zn, Mn, Co, Cd, Ni, and Pb) were determined in the sediment and the aboveground and belowground tissues of the selected macrophytes. Bioaccumulation factor (BF) and translocation factor (TF) were computed for each species. The sediment heavy metals concentrations of the three drains occurred in the following order: El-Westany > Amar > Omar-Beck. The concentrations of sediment heavy metals in the three drains were ordered as follows: Fe (438.45–615.17 mg kg−1) > Mn (341.22–481.09 mg kg−1) > Zn (245.08–383.19 mg kg−1) > Cu (205.41–289.56 mg kg−1) > Pb (31.49–97.73 mg kg−1) > Cd (13.97–55.99 mg kg−1) > Ni (14.36–39.34 mg kg−1) > Co (1.25–3.51 mg kg−1). The sediment exceeded the worldwide permissible ranges of Cu, Zn and Pb, but ranged within safe limits for Mn, Cd, Ni and Co. P. australis accumulated the highest concentrations of Fe, Co, Cd and Ni, while E. crassipes contained the highest concentrations of Cu, Zn, Mn, and Pb. Except for C. alopecuroides and Cu metal, the studied species had BF values greater than one for the investigated heavy metals. Nevertheless, the TFs of all species (except Cd in L. stolonifera) were less than one. Hence, the studied species are appropriate for accumulation, biomonitoring, and phytostabilization of the investigated metals.

This study investigated the growth response of Ranunculus sceleratus to pollution and its capacity to accumulate trace metals for its use as a phytoremediator in Lake Maruit, Egypt. Three basins (main basin, fish farm, and southwestern basin) representing the natural distribution of the plant as well as the pollution loads in the lake, were chosen for collecting plant and sediment samples. In each basin ten quadrats (0.5 m × 0.5 m), distributed equally along two sites, were selected for measuring growth parameters, nutrients, and trace metal concentration. The highest biomass of the shoot and root (610 and 236 g m −2 ) was recorded in the main basin and fish farm, respectively. R. sceleratus accumulated high concentrations of Cu and Pb (27.7 and 9.9 mg kg −1 ), while toxic concentration of Mn (2508.0 mg kg −1 ) in their roots compared to shoots. The bioaccumulation factor (BF) for the investigated metals was greater than one, and in the decreasing order: Ni (27.1) > Zn (20.0) > Cd (16.4) > Cu (7.7) > Mn (3.9) > Pb (3.6). The translocation factor of all analyzed trace metals was less than one. The ability of R. sceleratus to accumulate Mn, Ni, Cu, and Pb in its roots indicates the potential use of this species for phytostabilization of these metals (mainly Mn) in contaminated water bodies.

The aim of the present study was to develop an experimental system for an exploration of ethylene-dependent responses using intact growing Ranunculus sceleratus plants and to approbate the system for assessing the role of ethylene in salinity tolerance and ion accumulation. Plants were cultivated in sealed plastic containers in a modified gaseous atmosphere by introducing ethylene or 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. High humidity inside the containers induced a fast elongation of the leaf petioles of R. sceleratus. The effect was ethylene-dependent, as 1-MCP completely blocked it, but exogenous ethylene further promoted petiole elongation. Exogenous ethylene decreased (by 48%) but 1-MCP increased (by 48%) the Na+ accumulation in leaf blades of NaCl-treated plants. The experimental system was further calibrated with ethylene and silica xerogel, and the optimum concentrations were found for inducing leaf petiole elongation (10 μL L–1 ethylene) and preventing leaf petiole elongation (200 g silica xerogel per 24 L), respectively. The second experiment involved a treatment with NaCl in the presence of 1-MCP, ethylene, or 1-MCP + ethylene, both in normal and high air humidity conditions. In high humidity conditions, NaCl inhibited petiole elongation by 25% and ethylene treatment fully reversed this inhibition and stimulated elongation by 12% in comparison to the response of the control plants. Treatment with 1-MCP fully prevented this ethylene effect. In normal humidity conditions, NaCl inhibited petiole elongation by 20%, which was reversed by ethylene without additional elongation stimulation. However, 1-MCP only partially inhibited the ethylene effect on petiole elongation. In high humidity conditions, ethylene inhibited Na+ accumulation in NaCl-treated plants by 14%, but 1-MCP reversed this effect. In conclusion, the stimulation of endogenous ethylene production in R. sceleratus plants at a high air humidity or in flooded conditions reverses the inhibitory effect of salinity on plant growth and concomitantly inhibits the accumulation of Na+ in tissues. R. sceleratus is a highly promising model species for use in studies regarding ethylene-dependent salinity responses and ion accumulation potential involving the manipulation of a gaseous environment.