Andrzej Tretyn

Sequencing technologies and genome sequencing

The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of

The chemical characteristic and distribution of brassinosteroids in plants.

1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research.

Acetylcholine in plants : presence, metabolism and mechanism of action

Brassinosteroids represent a class of plant hormones with high-growth promoting activity. They are found at low levels in pollen, anthers, seeds, leaves, stems, roots, flowers, grain, and young vegetative tissues throughout the plant kingdom. Brassinosteroids are a family of about 60 phytosteroids. The article gives a comprehensive survey on the hitherto known brassinosteroids isolated from plants. The chemical characteristic of brassinosteroids is also presented.

The role(s) of calcium ions in phytochrome action.

Acetylcholine (ACh) has been detected in representatives of many taxonomic groups throughout the plant kingdom. The site of its synthesis in plants is probably young leaves. In some plant species choline acetyltransferase (ChAT) activity has been found. This enzyme showing properties similar to animal ChAT, probably participates in ACh synthesis from its precursors, choline and acetyl-Coenzyme A. Acetylcholinesterase (AChE) activity has also been found in many plant tissues. This enzyme decomposes ACh and exhibits properties similar to animal AChE. The presence of both ChAT and AChE in plant tissues suggests that ACh undergoes similar metabolism in plants as it does in animals. Exogenous ACh affects phytochrome-controlled plant growth and development. Mimicking red light (R), ACh stimulates adhesion of root tips to a glass surface and influences leaf movement and membrane permeability to ions. It also affects seed germination and plant growth. Moreover, ACh can modify some enzyme activity and the course of some metabolic processes in plants. Acetylcholine in the presence of calcium ions (Ca2+), like R stimulates swelling of protoplast isolated from etiolated wheat leaves. It is proposed that the primary mechanism of action of ACh in plant cells is via the regulation of membrane permeability to protons (H+), potassium ions (K+), sodium ions (Na+) and Ca2+.ZusammenfassungAcetylcholin (ACh) wurde in Vertretern vieler taxonomischer Gruppen des Pflanzreiches gefunden. Es wird wahrscheinlich inden jungen Blättern synthetisiert. In einigen Pflanzen hat man daneben Cholin-Acetyltransferase (ChAT)-Activität nachweisen können; dieses Enzym ziegt ähnliche Eigenschaften wie tierische ChAT und ist offenbar an der ACh-Synthese aus sienen Vorstufen Cholin und Acetyl-Coenzym A beteiligt. Acetylcholineesterase (AChE)-Activität wurde ebenfalls in vielen Pflanzengeweben gefunden; dieses Enzym spaltet ACh und ziegt ähnliche Eigenschaften wie tierische AChE. Die Anwesenheit von ChAT und AChE in pflanzlichem Gewebe läßt vermuten, daß ACh in Pflanzen einem ähnlichen Metabolismus unterliegt wie im tierischen System.Ähnlich wie Rotlicht stimuliert ACh die Anheftung von Wurzelspitzen an Glasoberflächen und beeinflußt Blattbewegung und Membranpermeabilität für Ionen; darüber hinaus beeinflußt es Samenkeimung und pflanzliches Wachstum. Des weiteren kann ACh Enzym-Aktivitäten modifizieren und dadurch den Ablauf einiher metabolischer Prozesse in Pflanzen. Schließlich stimuliert ACh in Gegenwart von Calcium-Ionen (Ca2+), ähnlich wie Rotlicht, das Schwellen von Protoplasten etiolierter Weizenblätter. Es wird vermutet, daß die Primärwirkung von ACh in Pflanzenzellen durch Regulation der Membranpermeabilität für Protonen (H+), Kaliumionen (K+), Natriumionen (Na+) und Ca2+ erfolgt.

Reactive oxygen species localization in roots of Arabidopsis thaliana seedlings grown under phosphate deficiency

Phytochrome, the red (R)*and far-red light (FR)absorbing morphogenetic photoreceptor, which occurs throughout the Plant Kingdom, was discovered by scientists at the US Department of Agriculture, Beltsville, MD. This year is the 40th anniversary of the prediction of the R/FR reversible pigment in plants and more than 30 years since its first spectroscopic detection. During the last four decades extensive progress has been made in understanding the molecular structure and function of phytochrome (for review see Furuya, 1987). Phytochrome genes have now been cloned for a few plant species (Quail et al . , 1987; Furuya, 1989; Sharrock and Quail, 1989). The photoregulation of genes, including the phytochrome gene itself, has been extensively studied (Quail et a / . , 1987; Nagy et a / . , 1988; Furuya, 1989; Tomizawa et a[., 1990). However, the molecular mechanism of phytochrome action is still obscure. Multiple response types could be an indication of different modes of action of the photoreceptor (Jordan et al., 1986; Kronenberg and Kendrick. 1986; Schafer et al., 1986; Furuya, 1989). One attractive hypothesis is that calcium ions (Ca2+) participate as a second messenger (Roux, 1984). During the last decade many papers have been published about the involvement of Ca” in the regulation of different phytochrome-regulated processes. Some o’f them were reviewed by Roux et 121. in 1986. Since that time

A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein

Arabidopsis plants responding to phosphorus (P) deficiency increase lateral root formation and reduce primary root elongation. In addition the number and length of root hairs increases in response to P deficiency. Here we studied the patterns of radical oxygen species (ROS) in the roots of Arabidopsis seedlings cultured on media supplemented with high or low P concentration. We found that P availability affected ROS distribution in the apical part of roots. If plants were grown on high P medium, ROS were located in the root elongation zone and quiescent centre. At low P ROS were absent in the elongation zone, however, their synthesis was detected in the primary root meristem. The proximal part of roots was characterized by ROS production in the lateral root primordia and in elongation zones of young lateral roots irrespective of P concentration in the medium. On the other hand, plants grown at high or low P differed in the pattern of ROS distribution in older lateral roots. At high P, the elongation zone was the primary site of ROS production. At low P, ROS were not detected in the elongation zone. However, they were present in the proximal part of the lateral root meristem. These results suggest that P deficiency affects ROS distribution in distal parts of Arabidopsis roots. Under P-sufficiency ROS maximum was observed in the elongation zone, under low P, ROS were not synthesized in this segment of the root, however, they were detected in the apical root meristem.

Exogenous auxin regulates H2O2 metabolism in roots of tomato (Lycopersicon esculentum Mill.) seedlings affecting the expression and activity of CuZn-superoxide dismutase, catalase, and peroxidase

Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.

The Mechanism of Calcium Action on Flower Induction in Pharbitis nil

The aim of this study was to test the effect of auxin treatment on selected parameters of the redox metabolism in roots. We found that auxin application results in a reduction in the H2O2 level in roots. The hormone stimulated CuZn-superoxide dismutase, but simultaneously increased the activities of catalase, cell wall bound ferulic acid peroxidase, and soluble peroxidase izoenzymes. The analysis of the expression of genes coding for the cytosolic izoform of CuZn-superoxide dismutase, catalase, and cell wall associated peroxidase (TPX 1) involved in cell wall stiffening and lignification revealed the stimulatory effect of exogenous auxin on the expression of the aforementioned genes. The enzyme activity and gene expression in the roots of control and auxin-treated plants were studied in daily intervals, during a 3-day-long growth cycle. The stimulatory effect of auxin on the enzymatic activity was transient with the highest stimulation observed on the second day of treatment. On the third day, the activities of the enzymes decreased. The maximal enzyme activities were preceded by a rise in gene expression. The increase in the level of CuZn-superoxide dismutase and catalase transcripts were detected after 1 day of auxin treatment. Then the expression of the aforementioned genes decreased. The period of auxin-dependent stimulation of the TPX 1 gene expression encompassed the first and the second day of treatment. Auxin stimulated CuZn-superoxide dismutase and catalase activities only in the distal zone of the root while peroxidase activity was increased by auxin in the distal as well as in the proximal parts of the organ.

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil

Summary Calcium ions, calcium ionophores A23187 and ionomycin, as well as caffeine, stimulated the flowering of Pharbitis nil when applied just before and 2 h after starting a subinductive, 14-h long dark period. Effectiveness of all the mentioned compounds decreased over successive hours and when used from 6 h after the onset of the dark period they had no effect or inhibited flowering. Intracellular calcium modulators, ryanodine and thapsigargicin, which are active in animal cells had no effect on flowering. These results suggest that an increase in free Ca 2+ before and during the first 2 h of the dark period increases the flowering responses, whereas after the 6th hour it decreases it. We postulate that the targets for calcium action are stomata, which are open before the dark period and remain closed during the first 4-5 h of the dark period. The significance of stomata in flower induction was confirmed in experiments with abscisic acid (ABA), a plant hormone which regulates stomatal movements. Treating the cotyledons of plants with ABA at the 8th and 10th hour of the dark period, resulted in plants with about 50% less flower buds than the control, however, this phytohormone had no effect on flowering when used before the subinductive dark period.

The role of light and polar auxin transport in root regeneration from hypocotyls of tomato seedling cuttings

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.