Features of Activity of the Phenylpropanoid Biosynthesis Pathway in Melanin-Accumulating Barley Grains.

Barley (Hordeum vulgare L.) grain pigmentation is caused by two types of phenolic compounds: anthocyanins (which are flavonoids) give a blue or purple color, and melanins (which are products of enzymatic oxidation and polymerization of phenolic compounds) give a black or brown color. Genes Ant1 and Ant2 determine the synthesis of purple anthocyanins in the grain pericarp, whereas melanins are formed under the control of the Blp1 gene in hulls and pericarp tissues. Unlike anthocyanin synthesis, melanin synthesis is poorly understood. The objective of the current work was to reveal features of the phenylpropanoid biosynthesis pathway functioning in melanin-accumulating barley grains. For this purpose, comparative transcriptomic and metabolomic analyses of three barley near-isogenic lines accumulating anthocyanins, melanins, or both in the grain, were performed. A comparative analysis of mRNA libraries constructed for three stages of spike development (booting, late milk, and early dough) showed transcriptional activation of genes encoding enzymes of the general phenylpropanoid pathway in all the lines regardless of pigmentation; however, as the spike matured, unique transcriptomic patterns associated with melanin and anthocyanin synthesis stood out. Secondary activation of transcription of the genes encoding enzymes of the general phenylpropanoid pathway together with genes of monolignol synthesis was revealed in the line accumulating only melanin. This pattern differs from the one observed in the anthocyanin-accumulating lines, where - together with the genes of general phenylpropanoid and monolignol synthesis pathways - flavonoid biosynthesis genes were found to be upregulated, with earlier activation of these genes in the line accumulating both types of pigments. These transcriptomic shifts may underlie the observed differences in concentrations of phenylpropanoid metabolites analyzed in the grain at a late developmental stage by high-performance liquid chromatography. Both melanin-accumulating lines showed an increased total level of benzoic acids. By contrast, anthocyanin-accumulating lines showed higher concentrations of flavonoids and p-coumaric and ferulic acids. A possible negative effect of melanogenesis on the total flavonoid content and a positive influence on the anthocyanin content were noted in the line accumulating both types of pigments. As a conclusion, redirection of metabolic fluxes in the phenylpropanoid biosynthesis pathway occurs when melanin is synthesized.

Discordant evolution of organellar genomes in peas (Pisum L.).

Plastids and mitochondria have their own small genomes, which do not undergo meiotic recombination and may have evolutionary fates different from each other and that of the nuclear genome. For the first time, we sequenced mitochondrial genomes of pea (Pisum L.) from 42 accessions mostly representing diverse wild germplasm from throughout the wild pea geographical range. Six structural types of the pea mitochondrial genome were revealed. From the same accessions, plastid genomes were sequenced. Phylogenetic trees based on the plastid and mitochondrial genomes were compared. The topologies of these trees were highly discordant, implying not less than six events of hybridisation between diverged wild peas in the past, with plastids and mitochondria differently inherited by the descendants. Such discordant inheritance of organelles could have been driven by plastid-nuclear incompatibility, which is known to be widespread in crosses involving wild peas and affects organellar inheritance. The topology of the phylogenetic tree based on nucleotide sequences of a nuclear gene, His5, encoding a histone H1 subtype, corresponded to the current taxonomy and resembled that based on the plastid genome. Wild peas (Pisum sativum subsp. elatius s.l.) inhabiting Southern Europe were shown to be of hybrid origin, resulting from crosses of peas related to those presently inhabiting the eastern Mediterranean in a broad sense. These results highlight the roles of hybridisation and cytonuclear conflict in shaping plant microevolution.

The pathogenic potential of the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice.

Parasitic food-borne diseases and chronic social stress are frequent attributes of day-to-day human life. Therefore, our aim was to model the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice. Histological examination of the liver revealed inflammation sites, pronounced periductal fibrosis, and cholangiofibrosis together with proliferation of bile ducts and hepatocyte dystrophy in the infected mice, especially in the stress-exposed ones. Simultaneously with liver pathology, we detected significant structural changes in the cerebral cortex. Immunohistochemical analysis of the hippocampus indicated the highest increase in numerical density of Iba 1-, IL-6-, iNOS-, and Arg1-positive cells in mice simultaneously subjected to the two adverse factors. The number of GFAP-positive cells rose during repeated social defeat stress, most strongly in the mice subjected to both infection and stress. Real-time PCR analysis showed that the expression of genes Aif1 and Il6 differed among the analysed brain regions (hippocampus, hypothalamus, and frontal cortex) and depended on the adverse factors applied. In addition, among the brain regions, there was no consistent increase or decrease in these parameters when the two adverse treatments were combined: (i) in the hippocampus, there was upregulation of Aif1 and no change in Il6 expression; (ii) in the hypothalamus, expression levels of Aif1 and Il6 were not different from controls; and (iii) in the frontal cortex, Aif1 expression did not change while Il6 expression increased. It can be concluded that a combination of two long-lasting adverse factors, O. felineus infection and repeated social defeat stress, worsens not only the hepatic but also brain state, as evidenced behaviorally by disturbances of the startle response in mice.

Cryptic divergences in the genus Pisum L. (peas), as revealed by phylogenetic analysis of plastid genomes.

Organellar genomes may shed light on complicated patterns of plant evolution at inter- and intraspecies level. Primary structure of plastid genomes sequenced in this study and taken from public databases was characterised and compared in 22 diverse, mostly wild representatives of the genus Pisum (peas). Phylogenetic trees reconstructed via Bayesian approach on the basis of entire plastid genomes resembled those reconstructed on the basis of a nuclear gene His5 coding for a minor histone H1 subtype. They reveal Pisum fulvum as an early divergence of the genus but do not support other taxonomical subdivisions. The positions of three accessions, classified as P. sativum subsp. elatius (the wild subspecies of the common pea), appeared quite unexpected. On the entire plastid genome tree, two accessions, from the Black Sea area of Turkey and Georgia, clustered with representatives of another species, P. fulvum, while the other, from Greece, was the first divergence of the P. sativum branch. We suppose these unusual plastid genomes to be ancient lineages ascending to a 'missing link' between P. fulvum and P. sativum, represented by accession Pe 013 from Turkey. Accessions with common pea appearance but deeply diverged plastids could occur through occasional crossing of diverged pea lines in the past and biparental plastid inheritance, both events being possible in peas.

Metabolic pathways and genes identified by RNA-seq analysis of barley near-isogenic lines differing by allelic state of the Black lemma and pericarp (Blp) gene.

BACKGROUND: Some plant species have 'melanin-like' black seed pigmentation. However, the chemical and genetic nature of this 'melanin-like' black pigment have not yet been fully explored due to its complex structure and ability to withstand almost all solvents. Nevertheless, identification of genetic networks participating in trait formation is key to understanding metabolic processes involved in the expression of 'melanin-like' black seed pigmentation. The aim of the current study was to identify differentially expressed genes (DEGs) in barley near-isogenic lines (NILs) differing by allelic state of the Blp (black lemma and pericarp) locus. RESULTS: RNA-seq analysis of six libraries (three replicates for each line) was performed. A total of 957 genome fragments had statistically significant changes in expression levels between lines BLP and BW, with 632 fragments having increased expression levels in line BLP and 325 genome fragments having decreased expression. Among identified DEGs, 191 genes were recognized as participating in known pathways. Among these were metabolic pathways including 'suberin monomer biosynthesis', 'diterpene phytoalexins precursors biosynthesis', 'cutin biosynthesis', 'cuticular wax biosynthesis', and 'phenylpropanoid biosynthesis, initial reactions'. Differential expression was confirmed by real-time PCR analysis of selected genes. CONCLUSIONS: Metabolic pathways and genes presumably associated with black lemma and pericarp colour as well as Blp-associated resistance to oxidative stress and pathogens, were revealed. We suggest that the black pigmentation of lemmas and pericarps is related to increased level of phenolic compounds and their oxidation. The effect of functional Blp on the synthesis of ferulic acid and other phenolic compounds can explain the increased antioxidant capacity and biotic and abiotic stress tolerance of black-grained cereals. Their drought tolerance and resistance to diseases affecting the spike may also be related to cuticular wax biosynthesis. In addition, upregulated synthesis of phytoalexins, suberin and universal stress protein (USP) in lemmas and pericarps of the Blp carriers may contribute to their increased disease resistance. Further description of the DEGs haplotypes and study of their association with physiological characteristics may be useful for future application in barley pre-breeding.

Efficient inhibition of influenza A viral replication in cells by deoxyribozymes delivered by nanocomposites.

Nucleic-acid-based drugs are a promising class of novel therapeutics; however, their use in medicine is widely limited because of insufficient delivery into cells. This article proposes a new delivery strategy of nucleic acid fragments into cells as components of TiO2-based nanocomposites. For the first time, unmodified Dz molecules were non-covalently immobilized on TiO2 nanoparticles precovered with polylysine (TiO2•PL) with the formation of (TiO2•PL)•Dz nanocomposites. DNAzymes in the proposed nanocomposites were shown to retain their ability to cleave the RNA target in a cell-free system with the same selectivity as unbound Dz molecules. It was shown by confocal laser microscopy that the fluorescein-labelled (TiO2•PL)•DzFlu nanocomposites penetrate into eukaryotic cells, where DzFlu is internalized in the cytoplasm and predominantly in nuclei. Delivery of deoxyribozymes into cells in the proposed nanocomposites permits very efficient interactions with RNA targets inside cells. This was demonstrated by an example of inhibition of H5N1 influenza A virus replication (inhibition by a factor of ca. 3000). This effect was one order of magnitude higher than with using lipofectamine as the transfection agent. The proposed (TiO2•PL)•Dz nanocomposites demonstrated high antiviral activity and are thus potent as nucleic-acid-based drugs.

SiO2 nanoparticles as platform for delivery of 3'-triazole analogues of AZT-triphosphate into cells.

A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO2 nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO2∼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO2∼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO2∼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO2∼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations.

Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

SiO2 nanoparticles as platform for delivery of nucleoside triphosphate analogues into cells.

A system for delivery of analogues of 2'-deoxyribonucleoside triphosphate (dNTP) based on SiO(2) nanoparticles was proposed. A simple and versatile method was developed for the preparation of SiO(2)-dNTP conjugates using the 'click'-reaction between premodified nanoparticles containing the azido groups and dNTP containing the alkyne-modified γ-phosphate group. The substrate properties of SiO(2)-dNTP were tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of the SiO(2)-dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate of polymerization with the use of SiO(2)-dNTP or common dNTP in case of HIV reverse transcriptase differed insignificantly. It was shown by confocal microscopy that the proposed SiO(2)-dNTP nanocomposites bearing the fluorescent label penetrate into cells and even into cellular nuclei.

Nanocomposites consisting of titanium dioxide nanoparticles and oligonucleotides.

The use of various nanoparticles is a promising way to solve the current problem of drug delivery in medicine and biology. Nanocomposites consisting of titanium dioxide and oligonucleotides noncovalently attached to nanoparticles through the polylysine linker (TiO2 x PL-DNA) have been designed to deliver of DNA fragments into cells. Three forms of TiO2 nanoparticles (amorphous, anatase, and brookite) were used for construction of nanocomposites. The size, morphology, and chemical composition of TiO2 nanoparticles and TiO2 x PL-DNA nanocomposites were characterized. DNA fragments in the proposed nanocomposites were shown to retain their ability to form complementary complexes. TiO2 x PL-DNA nanocomposites independently on the form of nanoparticles were shown by confocal microscopy to penetrate into HeLa cells without any transfection agents and physical impact. The presented type of nanocomposites can be applied in the thriving technology of drug delivery to achieve high therapeutic and biological efficacy.

High-affinity cation binding to organic cation transporter 1 induces movement of helix 11 and blocks transport after mutations in a modeled interaction domain between two helices.

Voltage-clamp fluorometry was performed with a cysteine-deprived mutant of rat organic cation transporter 1 (rOCT1) in which Phe483 in transmembrane alpha-helix (TMH) 11 close to the extracellular surface was replaced by cysteine and labeled with tetramethylrhodamine-6-maleimide. Potential-dependent fluorescence changes were observed that were sensitive to presence of substrates choline, tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP) and of the nontransported inhibitor tetrabutylammonium (TBuA). Using potential-dependent fluorescence changes as readout, one high-affinity binding site per substrate and two high-affinity binding sites for TBuA were identified in addition to the previously described single interaction sites. In a structure model of rOCT1 with an inward open cleft that was derived from a known crystal structure of lacY permease, Phe483 is close to Trp147 in TMH 2. In contrast, in a model with an outward open cleft these amino acids are far apart. After replacement of Phe483 or Trp147 by cysteine or serine, high-affinity binding of TBuA leads to inhibition of MPP or TEA uptake, whereas it has no effect on cation uptake by wild-type rOCT1. Coexisting high-affinity cation binding sites in organic cation transporters may collect low concentration xenobiotics and drugs; however, translocation including transitions between outward- and inward-oriented conformations may only be induced when a low-affinity cation binding site is loaded. We propose that cations bound to high-affinity sites may be translocated together with cations bound to low-affinity sites or that they may block the translocation mechanism.

Amino acids critical for substrate affinity of rat organic cation transporter 1 line the substrate binding region in a model derived from the tertiary structure of lactose permease.

To identify functionally relevant amino acids in the rat organic cation transporter 1 (rOCT1), 18 consecutive amino acids in the presumed fourth transmembrane alpha helix (TMH) were mutated and functionally characterized after expression in Xenopus laevis oocytes. After mutation of three amino acids on successive turns of the alpha helix, K(m) values for tetraethylammonium (TEA) and/or 1-methyl-4-phenylpyridinium (MPP) were decreased. After replacement of Trp218 by tyrosine (W218Y) and Tyr222 by leucine (Y222L), the K(m) values for both TEA and MPP were decreased. In mutants Y222F and T226A, only the K(m) values for TEA and MPP were decreased, respectively. The data suggest that amino acids Trp218 and Tyr222 participate in the binding of both TEA and MPP, whereas Thr226 is only involved in the binding of MPP. Using the crystal structure of the lactose permease LacY from Escherichia coli that belongs to the same major facilitator superfamily as rOCT1, we modeled the tertiary structure of the presumed 12 transmembrane alpha helices. The validity of the model was suggested because seven amino acids that have been shown to participate in the binding of cations by mutagenesis experiments [fourth TMH Trp218, Tyr222, and Thr226 (this work); 10th TMH Ala443, Leu447, and Gln448 (companion work in this issue of Molecular Pharmacology); 11th TMH Asp475 (previous report)] are located in one region surrounding a large cleft that opens to the intracellular side. The dimensions of TEA in comparison with the interacting amino acids in the modeled cleft suggest that more than one TEA molecule can bind in parallel to the modeled conformation of the transporter.

Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region.

The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are approximately 150 and approximately 4 microM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K(m) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 microM) uptake by corticosterone (24 +/- 4 microM) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 microM) uptake (5.3 +/- 1.7 microM). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.