Long-term organogenic callus cultivation of Ranunculus illyricus L.: a blueprint for sustainable ex situ conservation of the species in urban greenery.

The growing trend of introducing wild plant species into urban environments necessitates the identification of novel species adapted to prevailing conditions. A promising reservoir of such species may be xerothermic communities where Ranunculus illyricus occurs. This study aimed to establish a micropropagation protocol for R. illyricus using indirect organogenesis. The protocol includes initiation of culture from various explants, callus proliferation, shoot regeneration, multiplication, and concurrent rooting. Callus appeared on most types of vegetative explants tested, but stolons were considered the best due to their good availability, high disinfection (85%), and robust callus production (maximum increase - 363.1%). The growth rate of the callus fresh matter (CFM) obtained from stolons was calculated. Greater CFM was obtained on the medium with the supplemented picloram 8.0 mg L- 1 with kinetin 5.0 mg L- 1 and in second part of experiment on medium with the addition of 2,4-D (2,4-dichlorophenoxyacetic acid) 2.0 mg L- 1 alone or picloram 6.0 mg L- 1 with kinetin 8.0 mg L- 1. Shoot organogenesis was observed on macronutrients B5 (Gamborg medium), micronutrients MS (Murashige and Skoog) medium with the addition of 2.0 mg L- 1 IBA (indole-3-butyric acid) and 4.0 mg L- 1 BAP (6-benzylaminopurine). To document the process of callus differentiation, microscopic preparations were prepared. Subsequently, the regenerated plants underwent acclimatisation and their growth in an ex situ collection was monitored over three growing seasons. In particular, in vitro-origin plants exhibited developmental patterns similar to those of their seed-origin counterparts. The incorporation of R. illyricus into urban landscapes not only increases aesthetic appeal, but also ensures the preservation of valuable genetic resources for this rare species, potentially contributing to effective ex situ conservation in the future. This marks the first scientific report on in vitro cultures of R. illyricus.

In vitro and in vivo antitrypanosomal activity of the fresh leaves of Ranunculus Multifidus Forsk and its major compound anemonin against Trypanosoma congolense field isolate.

BACKGROUND: Animal trypanosomiasis is a major livestock problem due to its socioeconomic impacts in tropical countries. Currently used trypanocides are toxic, expensive, and the parasites have developed resistance to the existing drugs, which calls for an urgent need of new effective and safe chemotherapeutic agents from alternative sources such as medicinal plants. In Ethiopian traditional medicine fresh leaves of Ranunculus multifidus Forsk, are used for the treatment of animal trypanosomiasis. The present study aimed to evaluate the antitrypanosomal activity of the fresh leaves of R. multifidus and its major compound anemonin against Trypanosoma congolense field isolate. METHODS: Fresh leaves of R. multifidus were extracted by maceration with 80% methanol and hydro-distillation to obtain the corresponding extracts. Anemonin was isolated from the hydro-distilled extract by preparative TLC. For the in vitro assay, 0.1, 0.4, 2 and 4 mg/ml of the test substances were incubated with parasites and cessation or drop in motility of the parasites was monitored for a total duration of 1 h. In the in vivo assay, the test substances were administered intraperitoneally daily for 7 days to mice infected with Trypanosoma congolense. Diminazene aceturate and 1% dimethylsulfoxide (DMSO) were used as positive and negative controls, respectively. RESULTS: Both extracts showed antitrypanosomal activity although the hydro-distilled extract demonstrated superior activity compared to the hydroalcoholic extract. At a concentration of 4 mg/ml, the hydro-distilled extract drastically reduced motility of trypanosomes within 20 min. Similarly, anemonin at the same concentration completely immobilized trypanosomes within 5 min of incubation, while diminazene aceturate (28.00 mg/kg/day) immobilized the parasites within 10 min. In the in vivo antitrypanosomal assay, anemonin eliminates parasites at all the tested doses (8.75, 17.00 and 35.00 mg/kg/day) and prevented relapse, while in diminazene aceturate-treated mice the parasites reappeared on days 12 to 14. CONCLUSIONS: The current study demonstrated that the fresh leaves of R. multifidus possess genuine antitrypanosomal activity supporting the use of the plant for the treatment of animal trypanosomiasis in traditional medicine. Furthermore, anemonin appears to be responsible for the activity suggesting its potential as a scaffold for the development of safe and cost effective antitrypanosomal agent.

Full-length transcriptome analysis provides insights into flavonoid biosynthesis in Ranunculus japonicus.

Ranunculus japonicus Thunb. is a traditional Chinese herb. Plants in the genus Ranunculus are generally rich in flavonoids, which have antibacterial, anti-infective, and other pharmacological effects. However, owing to the lack of reference genomes, little is known about the flavonoid biosynthetic pathway in R. japonicus. In this study, PacBio isoform sequencing (PacBio iso-seq) and DNA nanoball sequencing (DNB-seq) were combined to build a full-length transcriptome database for three different tissues of R. japonicus. A total of 395,402 full-length transcripts were obtained, of which 308,474 were successfully annotated. A Kyoto Encyclopedia of Genes and Genomes analysis identified 29 differentially expressed genes encoding nine key enzymes for flavonoid biosynthesis. Correlation analysis indicated that flavanone 3-hydroxylase and flavonol synthase genes might have key roles in the accumulation of flavonoid substances in the different tissues of R. japonicus. The structures of chalcone synthase and chalcone isomerase enzymes were spatially modeled. Reverse-transcription quantitative PCR was used to verify gene expression levels of key enzymes associated with flavonoid biosynthesis. In addition, 22 MYB transcription factors involved in flavonoid biosynthesis and phenylpropanoid biosynthesis were discovered. The reliable transcriptomic data from this study provide genetic information about R. japonicus as well as insights into the molecular mechanism of flavonoid biosynthesis. The results also provide a basis for developing the medicinal value R. japonicus.

Untying Gordian knots: unraveling reticulate polyploid plant evolution by genomic data using the large Ranunculus auricomus species complex.

Speciation via hybridization and polyploidization is a major evolutionary force in plant evolution but is still poorly understood for neopolyploid groups. Challenges are attributed to high heterozygosity, low genetic divergence, and missing information on progenitors, ploidy, and reproduction. We study the large Eurasian Ranunculus auricomus species complex and use a comprehensive workflow integrating reduced-representation sequencing (RRS) genomic data to unravel reticulate evolution, genome diversity and composition of polyploids. We rely on 97 312 restriction site-associated DNA sequencing (RAD-Seq) loci, 576 targeted nuclear genes (48 phased), and 71 plastid regions derived from 78 polyploid apomictic taxa and four diploid and one tetraploid putative sexual progenitor species. We applied (phylo)genomic structure, network, and single nucleotide polymorphism (SNP)-origin analyses. Results consistently showed only 3-5 supported and geographically structured polyploid genetic groups, each containing extant sexual and one unknown progenitor species. Combined analyses demonstrated predominantly allopolyploid origins, each involving 2-3 different diploid sexual progenitor species. Young allotetraploids were characterized by subgenome dominance and nonhybrid SNPs, suggesting substantial post-origin but little lineage-specific evolution. The biodiversity of neopolyploid complexes can result from multiple hybrid origins involving different progenitors and substantial post-origin evolution (e.g. homoeologous exchanges, hybrid segregation, gene flow). Reduced-representation sequencing genomic data including multi-approach information is efficient to delimit shallow reticulate relationships.

Inhibitory activities of Ranunculus japonicus Thunb. ethanol extract against hepatitis B virus.

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation.

Detection of ranunculus mild mosaic virus in weed species in Japan.

Ranunculus (Ranunculus asiaticus L.) is one of the most popular cut flowers in Japan. However, the infection rate of ranunculus mild mosaic virus (RanMMV) in ranunculus plants has been gradually increasing during cultivation, suggesting that RanMMV may be transmitted from weeds to ranunculus plants in cultivation fields. In our survey, RanMMV in R. japonicus, R. tachiroei, R. cantoniensis, Geranium carolinianum, Vicia sativa, V. tetrasperma and V. hirsute in ranunculus fields and noncultivation regions in Japan was detected. Ranunculaceae weeds grow all year in cultivation fields, unlike R. asiaticus, indicating that these weeds may be a source of RanMMV infection. In addition, a pairwise comparison of CP genes between RanMMV isolates taken from R. asiaticus, R. japonicus, and R. tachiroei showed high nucleotide (98·1-100%) and amino acid (98·5-100%) identities. These results support the hypothesis that RanMMV may be transmitted between Ranunculaceae weeds and R. asiaticus plants. Thus, virus control should focus on removing host weeds from the cultivation fields.

Evolution of Transcriptomes in Early-Generation Hybrids of the Apomictic Ranunculus auricomus Complex (Ranunculaceae).

Hybridisation in plants may cause a shift from sexual to asexual seed formation (apomixis). Indeed, natural apomictic plants are usually hybrids, but it is still unclear how hybridisation could trigger the shift to apomixis. The genome evolution of older apomictic lineages is influenced by diverse processes such as polyploidy, mutation accumulation, and allelic sequence divergence. To disentangle the effects of hybridisation from these other factors, we analysed the transcriptomes of flowering buds from artificially produced, diploid F2 hybrids of the Ranunculus auricomus complex. The hybrids exhibited unreduced embryo sac formation (apospory) as one important component of apomixis, whereas their parental species were sexual. We revealed 2915 annotated single-copy genes that were mostly under purifying selection according to dN/dS ratios. However, pairwise comparisons revealed, after rigorous filtering, 79 genes under diversifying selection between hybrids and parents, whereby gene annotation assigned ten of them to reproductive processes. Four genes belong to the meiosis-sporogenesis phase (ASY1, APC1, MSP1, and XRI1) and represent, according to literature records, candidate genes for apospory. We conclude that hybridisation could combine novel (or existing) mutations in key developmental genes in certain hybrid lineages, and establish (together with altered gene expression profiles, as observed in other studies) a heritable regulatory mechanism for aposporous development.

Chemical Constituent Analysis of Ranunculus sceleratus L. Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

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.

Ranunculus ternatus Thunb extract attenuates renal fibrosis of diabetic nephropathy via inhibiting SMYD2.

CONTEXT: Ranunculus ternatus Thunb (Ranunculaceae), (RTT) is used clinically for the treatment of tuberculosis or as tumour adjuvant therapy, but its potential effect on diabetic nephropathy (DN) has not been studied. OBJECTIVE: To investigate the effect of RTT extract in renal fibrosis of DN. MATERIALS AND METHODS: C57BL/6 mice were randomly divided into four groups (n = 12). Diabetes mellitus (DM) mice were induced by streptozotocin (STZ, 55 mg/kg/day) for five consecutive days and treated by RTT extract (2 g/kg). Afterward, blood glucose, HE and Masson staining were assayed. The expression levels of Vimentin, ɑ-SMA, TNF-ɑ, NF-κB p-p65, NF-κB p65, SMYD2, H3K36me3, H3K4me3 were determined by western blots. Firbronectin was respectively assayed by western blot and immunofluorescent staining. RESULTS: RTT extract significantly ameliorated renal injury and renal fibrosis in the renal tissue of STZ-induced diabetic mice as demonstrated by the decreased expression level of Fibronectin (65%), Vimentin and α-SMA (75% & 53%). In addition, the levels of TNF-α (57%), NF-κB p-p65 and NF-κB p65 (35% & 25%) were elevated in the DN mice. Importantly, these were alleviated after RTT extract treatment. Moreover, we observed that the protein levels of SMYD2 (30%), H3K36me3 and H3K4me3 (53% & 75%) were reduced in DN mice after treatment with RTT extract. DISCUSSION AND CONCLUSIONS: RTT extract mediates antifibrotic effects and anti-inflammatory responses in STZ-induced DN mainly through suppressing SMYD2 activation and H3K36me3 and H3K4me3 protein expression. RTT extract might have therapeutic potential against high glucose-induced nephropathy.

Moving beyond assumptions: Polyploidy and environmental effects explain a geographical parthenogenesis scenario in European plants.

Geographical parthenogenesis (GP) describes the phenomenon that apomicts tend to have larger distribution areas and/or occur at higher altitudes or latitudes compared to sexual relatives. However, the complex effects of genome-wide heterozygosity, ploidy, reproduction mode (sexual vs. apomictic), and environment shaping GP of plants are still not well understood. We ascertained ploidy and reproduction mode by flow cytometry of 221 populations, and added genomic RADseq data (maximum 33,165 loci) of 80 taxa of the Ranunculus auricomus polyploid plant complex in temperate Europe. We observed 7% mainly diploid sexual, 28% facultative apomictic (mean sexuality 7.1%), and 65% obligate apomictic populations. Sexuals occupied a more southern, smaller distribution area, whereas apomicts expanded their range to higher latitudes. Within the complex, we detected three main genetic clusters and highly reticulate relationships. A genetically-informed path analysis using GLMMs revealed several significant relationships. Sexuality of populations (percent of sexual seeds) was higher in diploids compared to polyploids, associated with more petals, and similar between forests and open habitats. In contrast to other apomictic plant complexes, sexuality was mainly positively correlated to solar radiation and isothermality, which fits the southern distribution. We found up to three times higher heterozygosity in polyploids compared to diploids, and generally more heterozygous individuals in forests compared with open habitats. Interestingly, we revealed a previously unknown positive association between heterozygosity and temperature seasonality, suggesting a higher resistance of polyploids to more extreme climatic conditions. We provide empirical evidence for intrinsic and extrinsic factors shaping the GP pattern in a polyploid plant complex.

A molecular basis behind heterophylly in an amphibious plant, Ranunculus trichophyllus.

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.

In Vitro Antileishmanial and Antischistosomal Activities of Anemonin Isolated from the Fresh Leaves of Ranunculus multifidus Forsk.

Leishmaniasis and schistosomiasis are neglected tropical diseases (NTDs) infecting the world's poorest populations. Effectiveness of the current antileishmanial and antischistosomal therapies are significantly declining, which calls for an urgent need of new effective and safe drugs. In Ethiopia fresh leaves of Ranunculus multifidus Forsk. are traditionally used for the treatment of various ailments including leishmaniasis and eradication of intestinal worms. In the current study, anemonin isolated from the fresh leaves of R. multifidus was assessed for its in vitro antileishmanial and antischistosomal activities. Anemonin was isolated from the hydro-distilled extract of the leaves of R. multifidus. Antileishmanial activity was assessed on clinical isolates of the promastigote and amastigote forms of Leishmania aethiopica and L. donovani clinical isolates. Resazurin reduction assay was used to determine antipromastigote activity, while macrophages were employed for antiamastigote and cytotoxicity assays. Antischistosomal assays were performed against adult Schistosoma mansoni and newly transformed schistosomules (NTS). Anemonin displayed significant antileishmanial activity with IC50 values of 1.33 nM and 1.58 nM against promastigotes and 1.24 nM and 1.91 nM against amastigotes of L. aethiopica and L. donovani, respectively. It also showed moderate activity against adult S. mansoni and NTS (49% activity against adult S. mansoni at 10 µM and 41% activity against NTS at 1 µM). The results obtained in this investigation indicate that anemonin has the potential to be used as a template for designing novel antileishmanial and antischistosomal pharmacophores.

In Vivo Antimalarial Activity of Leaf Extracts and a Major Compound Isolated from Ranunculus multifidus Forsk.

The leaves of Ranunculus multifidus Forsk. are traditionally used for the treatment of malaria in several African countries. In the present study, 80% methanol (RM-M) and hydrodistilled (RM-H) extracts of fresh leaves from R. multifidus and its major constituent anemonin were tested for their in vivo antimalarial activity against Plasmodium berghei in mice. Anemonin was also tested for its in vitro antimycobacterial activity against Mycobacterium smegmatis and M. abscessus in a microbroth dilution assay, and bacterial growth was analyzed by OD measurement. The isolation of anemonin from RM-H was carried out using preparative thin layer chromatography (PTLC). The chemical structures of anemonin and its hydrolysis product were elucidated using spectroscopic methods (HR-MS; 1D and 2D-NMR). Results of the study revealed that both RM-M and RM-H were active against P. berghei in mice, although the latter demonstrated superior activity (p < 0.001), as compared to the former. At a dose of 35.00 mg/kg/day, RM-H demonstrated a chemosuppression value of 70% in a 4-day suppressive test. In a 4-day suppressive, Rane's and prophylactic antimalarial tests, anemonin showed median effective doses (ED50s) of 2.17, 2.78 and 2.70 µM, respectively. However, anemonin did not inhibit the growth of M. smegmatis and M. abscessus.

The relation of meiotic behaviour to hybridity, polyploidy and apomixis in the Ranunculus auricomus complex (Ranunculaceae).

BACKGROUND: Hybridization and polyploidization are powerful evolutionary factors that are associated with manifold developmental changes in plants such as irregular progression of meiosis and sporogenesis. The emergence of apomixis, which is asexual reproduction via seeds, is supposed to be connected to these factors and was often regarded as an escape from hybrid sterility. However, the functional trigger of apomixis is still unclear. Recently formed di- and polyploid Ranunculus hybrids, as well as their parental species were analysed for their modes of mega- and microsporogenesis by microscopy. Chromosomal configurations during male meiosis were screened for abnormalities. Meiotic and developmental abnormalities were documented qualitatively and collected quantitatively for statistical evaluations. RESULTS: Allopolyploids showed significantly higher frequencies of erroneous microsporogenesis than homoploid hybrid plants. Among diploids, F2 hybrids had significantly more disturbed meiosis than F1 hybrids and parental plants. Chromosomal aberrations included laggard chromosomes, chromatin bridges and disoriented spindle activities. Failure of megasporogenesis appeared to be much more frequent in than of microsporogenesis is correlated to apomixis onset. CONCLUSIONS: Results suggest diverging selective pressures on female and male sporogenesis, with only minor effects of hybridity on microsporogenesis, but fatal effects on the course of megasporogenesis. Hence, pollen development continues without major alterations, while selection will favour apomixis as alternative to the female meiotic pathway. Relation of investigated errors of megasporogenesis with the observed occurrence of apospory in Ranunculus hybrids identifies disturbed female meiosis as potential elicitor of apomixis in order to rescue these plants from hybrid sterility. Male meiotic disturbance appears to be stronger in neopolyploids than in homoploid hybrids, while disturbances of megasporogenesis were not ploidy-dependent.

Phylogenomics unravels Quaternary vicariance and allopatric speciation patterns in temperate-montane plant species: A case study on the Ranunculus auricomus species complex.

The time frame and geographical patterns of diversification processes in European temperate-montane herbs are still not well understood. We used the sexual species of the Ranunculus auricomus complex as a model system to understand how vicariance versus dispersal processes in the context of Pleistocene climatic fluctuations have triggered speciation in temperate-montane plant species. We used target enrichment sequence data from about 600 nuclear genes and coalescent-based species tree inference methods to resolve phylogenetic relationships among the sexual taxa of the complex. We estimated absolute divergence times and, using ancestral range reconstruction, we tested if speciation was enhanced by vicariance or by dispersal processes. Phylogenetic relationships among taxa were fully resolved with some incongruence in the position of the tetraploid R. marsicus. Speciation events took place in a very short time at the end of the Mid-Pleistocene Transition (830-580 thousand years ago [ka]). A second wave of intraspecific geographical differentiation occurred at the end of the Riss glaciation or during the Eemian interglacial between 200 and 100 ka. Ancestral range reconstruction suggests a widespread European ancestor of the R. auricomus complex. Vicariance has triggered allopatric speciation in temperate-montane plant species during the climatic deterioration that occurred during the last phase of the Mid-Pleistocene Transition. Vegetation restructuring from forest into tundra could have confined these forest species into isolated glacial macro- and microrefugia. During subsequent warming periods, range expansions of these species could have been hampered by apomictic derivatives and by other congeneric competitors in the same habitat.

The role of light quality of photoperiodic lighting on photosynthesis, flowering and metabolic profiling in Ranunculus asiaticus L.

Photoperiodic light quality affects flowering of long day plants, by influencing the phytochrome photoequilibria (PPE) at plant level; however, the most effective light spectrum to promote flowering is still unknown for most of the flower crops. We evaluated the influence of light spectrum of three light sources, with different induced PPE, on photosynthesis, metabolic profiling, plant growth and flowering in two hybrids of Ranunculus asiaticus L., MBO (early flowering) and MDR (medium earliness). Three photoperiodic treatments were compared to natural day length (NL): white fluorescent light (PPE 0.84), light emitting diodes (LEDs) with red:far red (R:FR) light at 3:1 ratio (PPE, 0.84) and LEDs with R:FR light at 1:3 ratio (PPE 0.63). Under natural light, net photosynthesis was higher in MDR than in MBO, while photochemistry was similar in the hybrids. Compared to NL, photoperiodic treatments did not affect net photosynthesis, while they promoted the quantum yield of PSII and reduced the non-photochemical quenching. Under NL, plant growth was greater in MBO, while flowering started earlier in MDR and flowers characteristics were similar in the hybrids. Despite the greater sensitivity of MDR plants in terms of metabolism, photoperiodic lighting improved plant growth and reduced the flowering time only in MBO, with a stronger effect under R:FR 3:1 light. MDR plants were characterized by higher soluble sugars, polyphenols, photosynthetic pigments and proteins, while MBO plants by higher starch and amino acid content. The morphological effects of photoperiodic light quality and the hybrid-specific response should be taken into account to optimize lighting protocols in commercial farms.

Winter Nights during Summer Time: Stress Physiological Response to Ice and the Facilitation of Freezing Cytorrhysis by Elastic Cell Wall Components in the Leaves of a Nival Species.

Ranunculus glacialis grows and reproduces successfully, although the snow-free time period is short (2-3 months) and night frosts are frequent. At a nival site (3185 m a.s.l.), we disentangled the interplay between the atmospheric temperature, leaf temperatures, and leaf freezing frequency to assess the actual strain. For a comprehensive understanding, the freezing behavior from the whole plant to the leaf and cellular level and its physiological after-effects as well as cell wall chemistry were studied. The atmospheric temperatures did not mirror the leaf temperatures, which could be 9.3 °C lower. Leaf freezing occurred even when the air temperature was above 0 °C. Ice nucleation at on average -2.6 °C started usually independently in each leaf, as the shoot is deep-seated in unfrozen soil. All the mesophyll cells were subjected to freezing cytorrhysis. Huge ice masses formed in the intercellular spaces of the spongy parenchyma. After thawing, photosynthesis was unaffected regardless of whether ice had formed. The cell walls were pectin-rich and triglycerides occurred, particularly in the spongy parenchyma. At high elevations, atmospheric temperatures fail to predict plant freezing. Shoot burial prevents ice spreading, specific tissue architecture enables ice management, and the flexibility of cell walls allows recurrent freezing cytorrhysis. The peculiar patterning of triglycerides close to ice rewards further investigation.

Epigenetic Patterns and Geographical Parthenogenesis in the Alpine Plant Species Ranunculus kuepferi (Ranunculaceae).

Polyploidization and the shift to apomictic reproduction are connected to changes in DNA cytosine-methylation. Cytosine-methylation is further sensitive to environmental conditions. We, therefore, hypothesize that DNA methylation patterns would differentiate within species with geographical parthenogenesis, i.e., when diploid sexual and polyploid apomictic populations exhibit different spatial distributions. On natural populations of the alpine plant Ranunculus kuepferi, we tested differences in methylation patterns across two cytotypes (diploid, tetraploid) and three reproduction modes (sexual, mixed, apomictic), and their correlation to environmental data and geographical distributions. We used methylation-sensitive amplified fragment-length polymorphism (methylation-sensitive AFLPs) and scored three types of epiloci. Methylation patterns differed independently between cytotypes versus modes of reproduction and separated three distinct combined groups (2x sexual + mixed, 4x mixed, and 4x apomictic), with differentiation of 4x apomicts in all epiloci. We found no global spatial autocorrelation, but instead correlations to elevation and temperature gradients in 22 and 36 epiloci, respectively. Results suggest that methylation patterns in R. kuepferi were altered by cold conditions during postglacial recolonization of the Alps, and by the concomitant shift to facultative apomixis, and by polyploidization. Obligate apomictic tetraploids at the highest elevations established a distinct methylation profile. Methylation patterns reflect an ecological gradient rather than the geographical differentiation.

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Blood pressure lowering effects of Ranunculus scleratus Linn. in normal and fructose induced hypertensive rats and estimation of underlying mechanisms.

Ranunculus scleratus Linn. is used in folk medicine to treat hypertension. This study was aimed at providing validation to its traditional use and to explore underlying mechanisms of action. Effects of hydro-ethanolic crude extract of the plant and its fractions on blood pressure was evaluated using direct surgical method in normotensive and in fructose induced hypertensive rats. Various doses of crude extract, RSC, (5, 10, 20, 30mg/kg) and all fractions (3, 5, 10, 20mg/kg) were studied. Results suggested that aqueous fraction of R. scleratus (RSA) produced most pronounced effects at 10mg/kg in normotensive and at 20mg/kg in hypertensive animals. Underlying mechanisms, using various pharmacological antagonists were also elucidated. Results suggested the involvement of muscarinic receptor, angiotensin converting enzyme (ACE) inhibition, ganglionic block and nitric oxide (NO) release in presenting hypotensive response.