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The order of Cyanidiales comprises seven acido-thermophilic red microalgal species thriving in hot springs of volcanic origin characterized by extremely low pH, moderately high temperatures and the presence of high concentrations of sulphites and heavy metals that are prohibitive for most other organisms. Little is known about the physiological processes underlying the long-term adaptation of these extremophiles to such hostile environments. Here, we investigated the long-term adaptive responses of a red microalga Cyanidioschyzon merolae, a representative of Cyanidiales, to extremely high nickel concentrations. By the comprehensive physiological, microscopic and elemental analyses we dissected the key physiological processes underlying the long-term adaptation of this model extremophile to high Ni exposure. These include: (i) prevention of significant Ni accumulation inside the cells; (ii) activation of the photoprotective response of non-photochemical quenching; (iii) significant changes of the chloroplast ultrastructure associated with the formation of prolamellar bodies and plastoglobuli together with loosening of the thylakoid membranes; (iv) activation of ROS amelioration machinery; and (v) maintaining the efficient respiratory chain functionality. The dynamically regulated processes identified in this study are discussed in the context of the mechanisms driving the remarkable adaptability of C. merolae to extremely high Ni levels exceeding by several orders of magnitude those found in the natural environment of the microalga. The processes identified in this study provide a solid basis for the future investigation of the specific molecular components and pathways involved in the adaptation of Cyanidiales to the extremely high Ni concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Due to translocation heterozygosity for all chromosomes in the cell complement, the oyster plant (Tradescantia spathacea) forms a complete meiotic ring. It also shows Rabl-arrangement at interphase, featured by polar centromere clustering. We demonstrate that the pericentromeric regions of the oyster plant are homogenized in concert by three subtelomeric sequences: 45S rDNA, (TTTAGGG)n motif, and TSrepI repeat. The Rabl-based clustering of pericentromeric regions may have been an excellent device to combine the subtelomere-pericentromere sequence migration (via inversions) with the pericentromere-pericentromere DNA movement (via whole arm translocations) that altogether led to the concerted homogenization of all the pericentromeric domains by the subtelomeric sequences. We also show that the repetitive sequence landscape of interstitial chromosome regions contains many loci consisting of Arabidopsis-type telomeric sequence or of TSrepI repeat, and it is extensively heterozygous. However, the sequence arrangement on some chromosomal arms suggest segmental inversions that are fully or partially homozygous, a fact that could be explained if the inversions started to create linkages already in a bivalent-forming ancestor. Remarkably, the subterminal TSrepI loci reside exclusively on the longer arms that could be due to sharing sequences between similarly-sized chromosomal arms in the interphase nucleus. Altogether, our study spotlights the supergene system of the oyster plant as an excellent model to link complex chromosome rearrangements, evolution of repetitive sequences, and nuclear architecture., (© 2022. The Author(s).)

Tomato ( Solanum lycopersicum L.) is a vegetable frequently exposed to hypoxia stress induced either by being submerged, flooded or provided with limited oxygen in hydroponic cultivation systems. The purpose of the study was to establish the metabolic mechanisms responsible for overcoming hypoxia in two tomato accessions with different tolerance to this stress, selected based on morphological and physiological parameters. For this purpose, 3-week-old plants (plants at the juvenile stage) of waterlogging-tolerant (WL-T), i.e., POL 7/15, and waterlogging-sensitive (WL-S), i.e., PZ 215, accessions were exposed to hypoxia stress (waterlogging) for 7 days, then the plants were allowed to recover for 14 days, after which another 7 days of hypoxia treatment was applied. Root samples were collected at the end of each time-point and 2D-DIGE with MALDI TOF/TOF, and expression analyses of gene and protein-encoded alcohol dehydrogenase (ADH2) and immunolabelling of ADH were conducted. After collating the obtained results, the different responses to hypoxia stress in the selected tomato accessions were observed. Both the WL-S and WL-T tomato accessions revealed a high amount of ADH2, which indicates an intensive alcohol fermentation pathway during the first exposure to hypoxia. In comparison to the tolerant one, the expression of the adh2 gene was about two times higher for the sensitive tomato. Immunohistochemical analysis confirmed the presence of ADH in the parenchyma cells of the cortex and vascular tissue. During the second hypoxia stress, the sensitive accession showed a decreased accumulation of ADH protein and similar expression of the adh2 gene in comparison to the tolerant accession. Additionally, the proteome showed a greater protein abundance of glyceraldehyde-3-phosphate dehydrogenase in primed WL-S tomato. This could suggest that the sensitive tomato overcomes the oxygen limitation and adapts by reducing alcohol fermentation, which is toxic to plants because of the production of ethanol, and by enhancing glycolysis. Proteins detected in abundance in the sensitive accession are proposed as crucial factors for hypoxia stress priming and their function in hypoxia tolerance is discussed.

Light-emitting diodes (LEDs) and high-pressure sodium lamps (HPS) are among the most commonly used light sources for plant cultivation. The objective of this study was to evaluate the effect of two controlled-environment production systems differing in light sources on growth, photosynthetic activity, and secondary metabolism of common buckwheat. We hypothesized that LED light with the majority of red and blue waves would increase physiological and biochemical parameters compared to sunlight supplemented with HPS lamps. The experiment was performed in a phytotronic chamber (LEDs) and in a greenhouse (solar radiation supplemented with HPS lamps as a control). The effects were analyzed at the flowering phase with biometric measurements, leaf chlorophyll index, the kinetics of chlorophyll a fluorescence, content of soluble carbohydrates and phenolics in the leaves. Applied LED light decreased the biomass but stimulated the production of phenolics compared to control plants. In control plants, a positive correlation between flavonoid content and energy dissipation from photosystem II (DI o /CS m ) was found, while in plants under LEDs total pool of phenolic content correlated with this parameter and the quantum yield of electron transport (φ Ro and ψ Ro) was lower than that of the control, probably affecting buckwheat biomass., (© 2022. The Author(s).)

Fusarium culmorum is a worldwide, soil-borne plant pathogen. It causes diseases of cereals, reduces their yield, and fills the grain with toxins. The main direction of modern breeding is to select wheat genotypes the most resistant to Fusarium diseases. This study uses seedlings and plants at the anthesis stage to analyze total soluble carbohydrates, total and cell-wall bound phenolics, chlorophyll content, antioxidant activity, hydrogen peroxide content, mycotoxin accumulation, visual symptoms of the disease, and Fusarium head blight index (FHBi). These results determine the resistance of three durum wheat accessions. We identify physiological or biochemical markers of durum wheat resistance to F. culmorum . Our results confirm correlations between FHBi and mycotoxin accumulation in the grain, which results in grain yield decrease. The degree of spike infection (FHBi) may indicate accumulation mainly of deoxynivalenol and nivalenol in the grain. High catalase activity in the infected leaves could be considered a biochemical marker of durum sensitivity to this fungus. These findings allowed us to formulate a strategy for rapid evaluation of the disease severity and the selection of plants with higher level, or resistance to F. culmorum infection.

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS-a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters-selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software-VCF2CAPS-fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services., Competing Interests: The authors have declared that no competing interests exist.

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This work is the first attempt to evaluate suitability of Callitriche cophocarpa Sendtn. (water-starwort) to remove Cr under real-world conditions. Our earlier laboratory-scale studies demonstrated outstanding hyperaccumulation properties of this aquatic higher plant (macrophyte) toward chromium in solution. We introduced C. cophocarpa plants into the watershed with sediments heavily polluted (on average 1400 mg/kg d.w. of Cr) by a tannery. The plants grew vigorously and exhibited no physiological or anatomical disorders. Based on chemical fractionations of bottom sediments, we found low Cr bioavailability. The element was strongly associated with the sediments and could be classified into the following fractions (%): oxidizable III (68.2) > residual IV (28.8) > reducible II (1.6) > exchangeable I (1.4). Despite this, Cr content in plant organs at the contaminated sites was 33 up to 83 times greater than in the control leaf/stem and roots, respectively. Altering redox potential during, i.e., sediment deposition on land may change chemical forms of bound metals in a solid phase, and thus further increase Cr phytoextraction by plants. With this in mind, we concluded that the species, being an outstanding Cr accumulator under laboratory conditions, can be useful in the reclamation of Cr-polluted sediments under controlled, oxidizing conditions.

Cell immobilization within nano-thin polymeric shells can provide an optimal concentration of biological material in a defined space and facilitate its directional growth. Herein, polyelectrolyte membrane scaffolds were constructed using a layer-by-layer approach to determine the possibility of promoting improved growth of rat cortical neuronal cells. Membrane presence was confirmed by Fourier transform infrared spectroscopy, Zeta potential, and atomic force and scanning electron microscopy. Scaffold performance toward neuronal cell growth was assessed in vitro during a 14-day culture. Cell conditions were analyzed immunocytochemically. Furthermore, western blot and real-time PCR analyses were used to validate the presence of neuronal and glial cells on the scaffolds. We observed that alginate/chitosan, alginate/polylysine, and polyethyleneimine/chitosan scaffolds promote neuronal growth similarly to the control, poly-d-lysine/laminin. We conclude that membranes maintaining cell viability, integrity and immobilization in systems supporting neuronal regeneration can be applied in neurological disease or wound healing treatment. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 839-850, 2019., (© 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc.)

Objective: The inadequate efficiency of existing therapeutic anti-cancer regiments and the increase in the multidrug resistance of cancer cells underscore the need to investigate novel anticancer strategies. The induction of apoptosis in tumors by cytotoxic agents produced by pathogenic microorganisms is an example of such an approach. Nevertheless, even the most effective drug should be delivered directly to targeted sites to reduce any negative impact on other cells. Accordingly, the stabilized nanosystem (SNS) for active agent delivery to cancer cells was designed for further application in local anti-tumor therapy. A product of genetically modified Escherichia coli, listeriolysin O (LLO), was immobilized within the polyelectrolyte membrane (poly(ethylenimine)|hyaluronic acid) shells of 'LLO nanocarriers' coupled with the stabilizing element of natural origin., Methods and Results: The impact of LLO was evaluated in human leukemia cell lines in vitro. Correspondingly, the influence of the SNS and its elements was assessed in vitro. The viability of targeted cells was evaluated by flow cytometry. Visualization of the system structure was performed using confocal microscopy. The membrane shell applied to the nanocarriers was analyzed using atomic force microscopy and Fourier transform infrared spectroscopy techniques. Furthermore, the presence of a polyelectrolyte layer on the nanocarrier surface and/or in the cell was confirmed by flow cytometry. Finally, the structural integrity of the SNS and the corresponding release of the fluorescent solute listeriolysin were investigated., Conclusion: The construction of a stabilized system offers LLO release with a lethal impact on model eukaryotic cells. The applied platform design may be recommended for local anti-tumor treatment purposes., Competing Interests: The authors have declared that no competing interests exist.

Carious is the most frequent disease of mineralized dental tissues which might result in dental pulp inflammation and mortality. In such cases an endodontic treatment is the only option to prolong tooth functioning in the oral cavity; however, in the cases of severe pulpitis, especially when complicated with periodontal tissue inflammation, the endodontic treatment might not be enough to protect against tooth loss. Thus, keeping the dental pulp viable and/or possibility of the reconstruction of a viable dental pulp complex, appears to become a critical factor for carious and/or pulp inflammation treatment. The nowadays technologies, which allow handling dental pulp stem cells (DPSC), seem to bring us closer to the usage of dental stem cells for tooth tissues reconstruction. Thus, DPSC immobilized within nano-thin polymeric shells, allowing for a diffusion of produced factors and separation from bacteria, may be considered as a cover system supporting technology of dental pulp reconstruction. The DPSC were immobilized using a layer-by-layer technique within nano-thin polymeric shells constructed and modified by nanostructure involvement to ensure the layers stability and integrity as well as separation from bacterial cells. The cytotoxity of the material used for membrane production was assessed on the model of adherent cells. The performance of DPSC nano-coating was assessed in vitro. Membrane coatings showed no cytotoxicity on the immobilized cells. The presence of coating shell was confirmed with flow cytometry, atomic force microscopy and visualized with fluorescent microscopy. The transfer of immobilized DPSC within the membrane system ensuring cells integrity, viability and protection from bacteria should be considered as an alternative method for dental tissues transportation and regeneration.

The reported analyses were aimed at identification of the mitochondrial proteome features which were associated with cytoplasmic male sterility (CMS) in beets. The set of analyzed accessions included CMS, maintainer and restored lines. Mitochondrial preparations were subjected to blue-native electrophoresis followed either by in-gel activity assays or separation in denaturing conditions. The CMS condition was associated with decreased activity of complex V and enhancement of additional complexes with the ATPase activity. This was accompanied by accumulation of heptamer HSP60, preSATP6 and an increase in the fraction of the free ATP9 oligomer (not bound to complex V). The ATP9 effect was reversed upon fertility restoration., Biological Significance: The reported work provides one of very few comprehensive comparisons of the mitochondrial proteomes from cytoplasmic male-sterile (CMS) and male-fertile plants. It shows that in beets the CMS trait is associated with altered functioning of the mitochondrial ATP synthase complex. The presence of CMS-specific ATP synthase subcomplexes resembles deficiencies of this enzyme reported for mammalian cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Key Message: Petaloid cytoplasmic male-sterile carrots exhibit overexpression of the mitochondrial atp9 genes which is associated with specific features in organization and expression of these sequences. In carrots, the Sp-cytoplasm causes transformation of stamens into petal-like organs, while plants carrying normal N-cytoplasm exhibit normal flower morphology. Our work was aimed at characterization of distinct features both cytoplasms display with respect to organization and expression of the mitochondrial atp9 genes. We show that two carrot atp9 genes, previously reported as cytoplasm-specific, in fact occur in heteroplasmic condition. In the Sp-cytoplasm the atp9-1 version dominates over atp9-3, while in N-cytoplasmic plants this proportion is reversed. Herein, we also indicate the presence and recombination activity of a 130-/172-bp sequence repeat which likely shaped the present organization of carrot atp9 loci. Furthermore, cDNA sequence examination revealed that the atp9 open reading frames (ORFs) were C to U edited in 4 nucleotide positions. One of the editing events turns a glutamine triplet into the stop codon, thereby equalizing ORFs of atp9-1 and atp9-3. A certain fraction of partially edited molecules was identified-they all represented the atp9-3 sequence. In either Sp- or N-cytoplasmic plants multiple 5' transcript termini were observed. Of these, the ones mapping more distantly from the atp9 ORF were more pronounced in case of petaloid accessions. It was also shown that despite comparable copy number of the genomic atp9 sequences, the level of the respective mRNAs was approximately 3 times higher in case of petaloid carrots. The latter fact corresponded to the elevated content of the ATP9 protein in plants carrying Sp-cytoplasm. The semi-fertile phenotype of such plants is associated with a drop in ATP9 accumulation.

Development of anticancer treatment strategies is ongoing considering still inadequate efficiency of existing anticancer therapeutics. Moreover, the lack of therapeutic agents selectivity against the tumor cells requires further investigations into novel anticancer strategies. The use of pathogenic microorganisms producing an oncolytic agent may be an approach for apoptotic therapy in cancer treatment. The purpose of this study was to investigate the targeting efficiency of Bacillus subtilis bacterial cells coated with modified polyelectrolyte shells applied to protect the bacterial cells from potential host immune response as well as to enhance the tumor-targeting efficiency. The shells were modified with transferrin to increase affinity toward the target tumor cells. The impact of bacterial cells coated with unmodified or modified nanothin shells on human leukemia cells was evaluated in vitro. It was observed that the bacterial cells coated with modified shells with incorporated transferrin exhibited stronger lethal impact on leukemia cells as compared to bacterial cells with unmodified shell coating. Applied modified membrane conformation allowing for functioning of encapsulated microorganisms may find potential use in local antitumor treatment purposes., (© 2013 Wiley Periodicals, Inc.)

The polymeric permiselective membranes application for immunoisolation of cells separating the transplanted cells from the host immunological system may eliminate immunosuppressive therapy during transplantation. The suitability of polyelectrolyte modified nanocoatings for immunoisolation of cells was assessed. The polymeric shells modified with incorporated fullerene derivate were applied for encapsulation of human T-lymphocyte cell line Jurkat or rat pancreatic islets of Langerhans using layer-by-layer technique. Hydroxylated fullerene was incorporated to the polyelectrolyte shell for hydrophility increase as well as for layer stability improvement. Evaluation with AFM, FTIR, fluorescence microscopy confirmed the nanocoating presence on the encapsulated cells. It was observed that polylysine-polyethyleneimine membrane with incorporated fullerenol allowed for encapsulated cells functioning in vitro. Membrane conformation applied for encapsulation of pancreatic rat islets allowed for glucose level decline during xenotransplantation into mice. The elaborated nanocoating may be recommended as the possible alternative to the space consuming microencapsulation for biomedical purposes.

Transfer of DNA between different compartments of the plant cell, i.e., plastid, mitochondrion and nucleus, is a well-known phenomenon in plant evolution. Six directions of inter-compartmental DNA migration are possible in theory, however only four of them have been previously reported. These include frequent cases of mitochondrion and plastid to nucleus transfer, plastid to mitochondrion transfer, and rare nucleus to mitochondrion migrations. The connection between the plastid and mitochondrial genomes in flowering plants has been viewed as a one way road. Contrary to these observations we found that a sequence widespread in the carrot mitochondrial genome, designated as Dc MP, was transferred to the plastid genome of a carrot ancestor. Interestingly, Dc MP was integrated into a tRNA promoter of the plastid trnV gene, replacing the original promoter sequence. The rearrangement of the plastid genome is specific for carrot and closely related species belonging Scandiceae clade. The structure of the sequence and the presence of a 6 nt target site duplication led us to speculate that the transfer was a result of a transposition event of a non-LTR retrotransposon. These findings open interesting questions about the evolution of organellar genomes and mobile genetic elements and provide a useful plastid marker to phylogenetically delineate species relationships within the Scandiceae clade.

Background: Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA., Results: Sequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome., Conclusions: This study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

Encapsulation of cells in polymeric shells allows for separation of biological material from produced factors, which may find biotechnological and biomedical applications. Human T-lymphocyte cell line Jurkat as well as rat pancreatic islets were encapsulated using LbL technique within shells of polyelectrolyte modified by incorporation of biotin complexed with avidin to improve cell coating and to create the potential ability to elicit specific biochemical responses. The coating with nano-thin modified shells allowed for maintenance of the evaluated cells' integrity and viability during the 8-day culture. The different PE impact may be observed on different biological materials. The islets exhibited lower mitochondrial activity than the Jurkat cells. Nevertheless, coating of cells with polyelectrolyte modified membrane allowed for functioning of both model cell types: 10 μm leukemia cells or 150 μm islets during the culture. Applied membranes maintained the molecular structure during the culture period. The conclusion is that applied modified membrane conformation may be recommended for coating shells for biomedical purposes.

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia., (© The Author(s) 2011. This article is published with open access at Springerlink.com)

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A(3)-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.