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.

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.

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.