Agnieszka Zmienko

Deciphering priming-induced improvement of rapeseed (Brassica napus L.) germination through an integrated transcriptomic and proteomic approach

Rape seeds primed with -1.2 MPa polyethylene glycol 6000 showed improved germination performance. To better understand the beneficial effect of osmopriming on seed germination, a global expression profiling method was used to compare, for the first time, transcriptomic and proteomic data for osmoprimed seeds at the crucial phases of priming procedure (soaking, drying), whole priming process and subsequent germination. Brassica napus was used here as a model to dissect the process of osmopriming into its essential components. A total number of 952 genes and 75 proteins were affected during the main phases of priming and post-priming germination. Transcription was not coordinately associated with translation resulting in a limited correspondence between mRNAs level and protein abundance. Soaking, drying and final germination of primed seeds triggered distinct specific pathways since only a minority of genes and proteins were involved in all phases of osmopriming while a vast majority was involved in only one single phase. A particular attention was paid to genes and proteins involved in the transcription, translation, reserve mobilization, water uptake, cell cycle and oxidative stress processes.

Selection of Reference Genes for qPCR- and ddPCR-Based Analyses of Gene Expression in Senescing Barley Leaves

Leaf senescence is a tightly regulated developmental or stress-induced process. It is accompanied by dramatic changes in cell metabolism and structure, eventually leading to the disintegration of chloroplasts, the breakdown of leaf proteins, internucleosomal fragmentation of nuclear DNA and ultimately cell death. In light of the global and intense reorganization of the senescing leaf transcriptome, measuring time-course gene expression patterns in this model is challenging due to the evident problems associated with selecting stable reference genes. We have used oligonucleotide microarray data to identify 181 genes with stable expression in the course of dark-induced senescence of barley leaf. From those genes, we selected 5 candidates and confirmed their invariant expression by both reverse transcription quantitative PCR and droplet digital PCR (ddPCR). We used the selected reference genes to normalize the level of the expression of the following senescence-responsive genes in ddPCR assays: SAG12, ICL, AGXT, CS and RbcS. We were thereby able to achieve a substantial reduction in the data variability. Although the use of reference genes is not considered mandatory in ddPCR assays, our results show that it is advisable in special cases, specifically those that involve the following conditions: i) a low number of repeats, ii) the detection of low-fold changes in gene expression or iii) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.

Oncogenomic portals for the visualization and analysis of genome-wide cancer data

Somatically acquired genomic alterations that drive oncogenic cellular processes are of great scientific and clinical interest. Since the initiation of large-scale cancer genomic projects (e.g., the Cancer Genome Project, The Cancer Genome Atlas, and the International Cancer Genome Consortium cancer genome projects), a number of web-based portals have been created to facilitate access to multidimensional oncogenomic data and assist with the interpretation of the data. The portals provide the visualization of small-size mutations, copy number variations, methylation, and gene/protein expression data that can be correlated with the available clinical, epidemiological, and molecular features. Additionally, the portals enable to analyze the gathered data with the use of various user-friendly statistical tools. Herein, we present a highly illustrated review of seven portals, i.e., Tumorscape, UCSC Cancer Genomics Browser, ICGC Data Portal, COSMIC, cBioPortal, IntOGen, and BioProfiling.de. All of the selected portals are user-friendly and can be exploited by scientists from different cancer-associated fields, including those without bioinformatics background. It is expected that the use of the portals will contribute to a better understanding of cancer molecular etiology and will ultimately accelerate the translation of genomic knowledge into clinical practice.

Analysis of large mutations in BARD1 in patients with breast and/or ovarian cancer: the Polish population as an example

Only approximately 50% of all familial breast cancers can be explained by known genetic factors, including mutations in BRCA1 and BRCA2. One of the most extensively studied candidates for breast and/or ovarian cancer susceptibility is BARD1. Although it was suggested that large mutations may contribute substantially to the deleterious variants of BARD1, no systematic study of the large mutations in BARD1 has been performed. To further elucidate the role of large mutations in BARD1, we designed a multiplex ligation-dependent probe amplification (MLPA) assay and performed an analysis of 504 women with a familial breast and/or ovarian cancer and 313 patients with ovarian cancer. The investigation did not reveal any large mutations in the BARD1 gene. Although the analysis was not focused on identification of small mutations, we detected seven deleterious or potentially deleterious point mutations, which contribute substantially to the total number of BARD1 mutations detected so far. In conclusion, although we cannot exclude the presence of large mutations in BARD1, our study indicates that such mutations do not contribute substantially to the risk of breast and/or ovarian cancer. However, it has to be noted that our results may be specific to the Polish population.

From Accumulation to Degradation: Reprogramming Polyamine Metabolism Facilitates Dark-Induced Senescence in Barley Leaf Cells

The aim of this study was to analyze whether polyamine (PA) metabolism is involved in dark-induced Hordeum vulgare L. ‘Nagrad’ leaf senescence. In the cell, the titer of PAs is relatively constant and is carefully controlled. Senescence-dependent increases in the titer of the free PAs putrescine, spermidine, and spermine occurred when the process was induced, accompanied by the formation of putrescine conjugates. The addition of the anti-senescing agent cytokinin, which delays senescence, to dark-incubated leaves slowed the senescence-dependent PA accumulation. A feature of the senescence process was initial accumulation of PAs at the beginning of the process and their subsequent decrease during the later stages. Indeed, the process was accompanied by both enhanced expression of PA biosynthesis and catabolism genes and an increase in the activity of enzymes involved in the two metabolic pathways. To confirm whether the capacity of the plant to control senescence might be linked to PA, chlorophyll fluorescence parameters, and leaf nitrogen status in senescing barley leaves were measured after PA catabolism inhibition and exogenously applied γ-aminobutyric acid (GABA). The results obtained by blocking putrescine oxidation showed that the senescence process was accelerated. However, when the inhibitor was applied together with GABA, senescence continued without disruption. On the other hand, inhibition of spermidine and spermine oxidation delayed the process. It could be concluded that in dark-induced leaf senescence, the initial accumulation of PAs leads to facilitating their catabolism. Putrescine supports senescence through GABA production and spermidine/spermine supports senescence-dependent degradation processes, is verified by H2O2 generation.

A gene expression microarray for Nicotiana benthamiana based on de novo transcriptome sequence assembly.

BackgroundNicotiana benthamiana has been widely used in laboratories around the world for studying plant-pathogen interactions and posttranscriptional gene expression silencing. Yet the exploration of its transcriptome has lagged behind due to the lack of both adequate sequence information and genome-wide analysis tools, such as DNA microarrays. Despite the increasing use of high-throughput sequencing technologies, the DNA microarrays still remain a popular gene expression tool, because they are cheaper and less demanding regarding bioinformatics skills and computational effort.ResultsWe designed a gene expression microarray with 103,747 60-mer probes, based on two recently published versions of N. benthamiana transcriptome (v.3 and v.5). Both versions were reconstructed from RNA-Seq data of non-strand-specific pooled-tissue libraries, so we defined the sense strand of the contigs prior to designing the probe. To accomplish this, we combined a homology search against Arabidopsis thaliana proteins and hybridization to a test 244k microarray containing pairs of probes, which represented individual contigs. We identified the sense strand in 106,684 transcriptome contigs and used this information to design an Nb-105k microarray on an Agilent eArray platform. Following hybridization of RNA samples from N. benthamiana roots and leaves we demonstrated that the new microarray had high specificity and sensitivity for detection of differentially expressed transcripts. We also showed that the data generated with the Nb-105k microarray may be used to identify incorrectly assembled contigs in the v.5 transcriptome, by detecting inconsistency in the gene expression profiles, which is indicated using multiple microarray probes that match the same v.5 primary transcripts.ConclusionsWe provided a complete design of an oligonucleotide microarray that may be applied to the research of N. benthamiana transcriptome. This, in turn, will allow the N. benthamiana research community to take full advantage of microarray capabilities for studying gene expression in this plant. Additionally, by defining the sense orientation of over 106,000 contigs, we substantially improved the functional information on the N. benthamiana transcriptome. The simple hybridization-based approach for detecting the sense orientation of computationally assembled sequences can be used for updating the transcriptomes of other non-model organisms, including cases where no significant homology to known proteins exists.

Time course transcriptional profiling of senescing barley leaves

Cell senescence occurs as a part of developmental or stress-induced process. It is tightly regulated and involves a sequence of metabolic and structural alterations, eventually leading to cell death. Dark-induced leaf senescence is a useful model for studying senescence-related events. To facilitate the integration of physiological and molecular studies utilizing this model, we generated the microarray data set providing time course gene expression profiles in senescing barley leaves. Here, we describe the detailed procedures and data analysis scheme of our experiment. The entire data set (available at NCBI/GEO database under GSE62539) has been successively explored to find the genes differentially expressed during the senescence process as well as to identify genes with the invariant expression as reliable references for qPCR or ddPCR experiments.

MLPA-Based Analysis of Copy Number Variation in Plant Populations

Copy number variants (CNVs) are intraspecies duplications/deletions of large DNA segments (>1 kb). A growing number of reports highlight the functional and evolutionary impact of CNV in plants, increasing the need for appropriate tools that enable locus-specific CNV genotyping on a population scale. Multiplex ligation-dependent probe amplification (MLPA) is considered a gold standard in genotyping CNV in humans. Consequently, numerous commercial MLPA assays for CNV-related human diseases have been created. We routinely genotype complex multiallelic CNVs in human and plant genomes using the modified MLPA procedure based on fully synthesized oligonucleotide probes (90–200 nt), which greatly simplifies the design process and allows for the development of custom assays. Here, we present a step-by-step protocol for gene-specific MLPA probe design, multiplexed assay setup and data analysis in a copy number genotyping experiment in plants. As a case study, we present the results of a custom assay designed to genotype the copy number status of 12 protein coding genes in a population of 80 Arabidopsis accessions. The genes were pre-selected based on whole genome sequencing data and are localized in the genomic regions that display different levels of population-scale variation (non-variable, biallelic, or multiallelic, as well as CNVs overlapping whole genes or their fragments). The presented approach is suitable for population-scale validation of the CNV regions inferred from whole genome sequencing data analysis and for focused analysis of selected genes of interest. It can also be very easily adopted for any plant species, following optimization of the template amount and design of the appropriate control probes, according to the general guidelines presented in this paper.

RNA-Seq-based analysis of differential gene expression associated with hepatitis C virus infection in a cell culture.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver diseases. Unfortunately, the mechanisms of HCV infection-induced liver injury and host-virus interactions are still not well recognized. To better understand these processes we determined the changes in the host gene expression that occur during HCV infection of Huh-7.5 cells. As a result, we identified genes that may contribute to the immune and metabolic cellular responses to infection. Pathway enrichment analysis indicated that HCV induced an increased expression of genes involved in mitogen-activated protein kinases signaling, adipocytokine signaling, cell cycle and nitrogen metabolism. In addition, the enrichment analyses of processes and molecular functions revealed that the up-regulated genes were mainly implicated in the negative regulation of phosphorylation. Construction of the pathway-gene-process network enabled exploration of a much more complex landscape of molecular interactions. Consequently, several essential processes altered by HCV infection were identified: negative regulation of cell cycle, response to endoplasmic reticulum stress, response to reactive oxygen species, toll-like receptor signaling and pattern recognition receptor signaling. The analyses of genes whose expression was decreased upon HCV infection showed that the latter were engaged in the metabolism of lipids and amino acids. Moreover, we observed disturbance in the cellular antiviral defense. Altogether, our results demonstrated that HCV infection elicits host response that includes a very wide range of cellular mechanisms. Our findings significantly broaden the understanding of complex processes that accompany HCV infection. Consequently, they may be used for developing new host-oriented therapeutic strategies.

Small RNA fragments derived from multiple RNA classes – the missing element of multi-omics characteristics of the hepatitis C virus cell culture model

BackgroundA pool of small RNA fragments (RFs) derived from diverse cellular RNAs has recently emerged as a rich source of functionally relevant molecules. Although their formation and accumulation has been connected to various stress conditions, the knowledge on RFs produced upon viral infections is very limited. Here, we applied the next generation sequencing (NGS) to characterize RFs generated in the hepatitis C virus (HCV) cell culture model (HCV-permissive Huh-7.5 cell line).ResultsWe found that both infected and non-infected cells contained a wide spectrum of RFs derived from virtually all RNA classes. A significant fraction of identified RFs accumulated to similar levels as miRNAs. Our analysis, focused on RFs originating from constitutively expressed non-coding RNAs, revealed three major patterns of parental RNA cleavage. We found that HCV infection induced significant changes in the accumulation of low copy number RFs, while subtly altered the levels of high copy number ones. Finally, the candidate RFs potentially relevant for host-virus interactions were identified.ConclusionsOur results indicate that RFs should be considered an important component of the Huh-7.5 transcriptome and suggest that the main factors influencing the RF biogenesis are the RNA structure and RNA protection by interacting proteins. The data presented here significantly complement the existing transcriptomic, miRnomic, proteomic and metabolomic characteristics of the HCV cell culture model.