Miroslaw Kwasniewski

QuantPrime – a flexible tool for reliable high-throughput primer design for quantitative PCR

BackgroundMedium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.ResultsHere we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet http://www.quantprime.de/ or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%.ConclusionQuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays.

Molecular Cloning and Characterization of β-Expansin Gene Related to Root Hair Formation in Barley

Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the β-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley.

Accumulation of peroxidase-related reactive oxygen species in trichoblasts correlates with root hair initiation in barley.

Root hairs are an important model in studies of cell differentiation and development in higher plants. The function of NADPH oxidase-related reactive oxygen species (ROS) in root hair development has been reported extensively in studies on Arabidopsis. In this study, we investigated the mechanism of the initiation of root hair formation, mediated by the peroxidase-dependent production of the highly reactive hydroxyl radical in barley (Hordeum vulgare L.). The distribution of ROS, including the hydroxyl radical (OH) and superoxide (O(2)(-)) was assessed using hydroxyphenyl fluorescein and nitroblue tetrazolium chloride, respectively, in the roots of wild-type plants and two root-hair mutants: root-hairless (rhl1.a) and with root hair growth blocked at the primordium stage (rhp1.b). Peroxidase-dependent OH accumulation was linked to root hair initiation and growth in plants where root hair formation was at least initiated, whereas OH was not detectable in the epidermis of the root-hairless mutant rhl1.a. O(2)(-) distribution in the roots of rhl1.a and rhp1.b mutants was not impaired and did not influence the root hair phenotype. Peroxidase inhibitor treatments of wild-type roots dramatically reduced the ability of growing roots to form root hairs and thus phenocopied the root-hairless phenotype. Expression of two candidate peroxidase genes, HvPRX45 and HvPRX2, was analyzed and their possible role in root hair-specific production of hydroxyl radicals was discussed. We propose a model of a two-step, coordinated ROS formation process in root hair cells that involves root hair-specific peroxidase(s) and root hair-specific NADPH oxidase necessary for a proper root hair formation in barley.

iRootHair: a comprehensive root hair genomics database.

The specialized root epidermis cells of higher plants produce long, tubular outgrowths called root hairs. Root hairs play an important role in nutrient and water uptake, and they serve as a valuable model in studies of plant cell morphogenesis. More than 1,300 articles that describe the biological processes of these unique cells have been published to date. As new fields of root hair research are emerging, the number of new papers published each year and the volumes of new relevant data are continuously increasing. Therefore, there is a general need to facilitate studies on root hair biology by collecting, presenting, and sharing the available information in a systematic, curated manner. Consequently, in this paper, we present a comprehensive database of root hair genomics, iRootHair, which is accessible as a Web-based service. The current version of the database includes information about 153 root hair-related genes that have been identified to date in dicots and monocots along with their putative orthologs in higher plants with sequenced genomes. In order to facilitate the use of the iRootHair database, it is subdivided into interrelated, searchable sections that describe genes, processes of root hair formation, root hair mutants, and available references. The database integrates bioinformatics tools with a focus on sequence identification and annotation. iRootHair is a unique resource for root hair research that integrates the large volume of data related to root hair genomics in a single, curated, and expandable database that is freely available at www.iroothair.org.

Water-deficiency conditions differently modulate the methylome of roots and leaves in barley (Hordeum vulgare L.)

Highlight Characterization of barley methylome modulation in leaves and roots under water-deficiency stress and following rewatering with respect to organ-specific responses.

Chromosomal localization of a novel repetitive sequence in the Chenopodium quinoa genome.

In this study, a novel repetitive sequence pTaq10 was isolated from theTaq I digest of the genomic DNA of the pseudocerealChenopodium quinoa. Sequence analysis indicated that this 286-bp monomer is not homologous to any known retroelement sequence. FISH and Southern blot analysis showed that this sequence is characterized by an interspersed genomic organization. After FISH, hybridization signals were observed as small dots spread throughout all of the chromosomes. pTaq hybridization signals were excluded from 45S rRNA gene loci, but they partly overlapped with 5S rDNA loci. pTaq10 is not a species-specific sequence, as it was also detected inC. berlandieri.

Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells

We used comet‐fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24‐h post‐treatment recovery period allowed a partial reversibility of MH‐induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet‐FISH in plants may be a useful tool for environmental monitoring assessment. Environ. Mol. Mutagen., 2012.

AN OPTIMIZED METHOD FOR THE ISOLATION OF NUCLEI FROM CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent‐based cell lysis. The nuclei‐enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel‐based and non‐gel‐based proteomic approaches.

Cytomolecular Analysis of Ribosomal DNA Evolution in a Natural Allotetraploid Brachypodium hybridum and Its Putative Ancestors—Dissecting Complex Repetitive Structure of Intergenic Spacers

Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n = 30), which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n = 10) and B. stacei (2n = 20). Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs) from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic level.

Stimulatory Effect of Xenobiotics on Oxidative Electron Transport of Chemolithotrophic Nitrifying Bacteria Used as Biosensing Element

Electron transport chain (ETCh) of ammonium (AOB) and nitrite oxidizing bacteria (NOB) participates in oxidation of ammonium to nitrate (nitrification). Operation of ETCh may be perturbed by a range of water-soluble xenobiotics. Therefore, consortia of nitrifying bacteria may be used as a biosensor to detect water contamination. A surprising feature of this system is an increase of oxygen consumption, detected in the presence of certain inhibitors of ETCh. Thus, to shed light on the mechanism of this effect (and other differences between inhibitors) we monitored separately respiration of the bacteria of the first (AOB - Nitrosomonas) and second (NOB -Nitrobacter) stages of nitrification. Furthermore, we measured plasma membrane potential and the level of reduction of NAD(P)H. We propose a novel model of ETCh in NOB to explain the role of reverse electron transport in the stimulation of oxygen consumption (previously attributed to hormesis).