Pecinka lab:Research

Centre of Plant Structural and Functional Genomics Institute of Experimental Botany of the Czech Academy of Sciences, v. v. i. Šlechtitelů 31 Olomouc, CZ-783 71 Czech Republic Contact

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Plant Genome Biology Genetic information of a living organism is constantly exposed to internal and external DNA damaging factors, such as reactive oxygen species, solar UV radiation or cleavage by endogenous transposases (Britt, 1996; Kimura and Sakaguchi, 2006; Hakem, 2008; Lisch, 2013). Robust protection of genetic information may be particularly important for sedentary organisms with late separation of germline cells, such as plants. Therefore, controlling genome integrity by the repair of damaged DNA and epigenetic silencing of transposable elements are the key molecular functions needed for a long term plant survival (Britt, 1996; Kimura and Sakaguchi, 2006; Law and Jacobsen, 2010; Lisch, 2013). The goal of our laboratory is to understand the molecular mechanisms of plant genome protection and repair under ambient and stress conditions. Current Projects DNA damage repair mechanisms in plants – the key to genome integrity Mutagenic effects of solar UV-B radiation. Plants require sunlight for photosynthesis. However, UV-B (280-312 nm) may cause damage of membranes, proteins and nucleic acids. In DNA, UV-B introduces non-native bonds between two pyrimidine sites, which can result in mutations if not repaired. Although the key repair components are known, it remains unknown how many UV-B mutations are induced in meristems and transmitted to next generations by solar doses of UV-B radiation. We address this question in collaboration with the sun simulator facility at the Helmholtz Zentrum in Munich and Schneeberger lab at MPIPZ. This will elucidate what is the number and spectrum of mutations caused by UV-B radiation under 'natural' conditions and how does this contribute to plant genome evolution. Arabidopsis natural variation in response to DNA replication stress. Figure 2 DNA replication is regulated in developmental and environmental factors and is essential for cell division, expansion and differentiation. We analyzed survival of A. thaliana accessions in response to DNA replication stress induced by hydroxyurea (HU). A combination of genome-wide association studies and quantitative trait locus mapping revealed candidate genomic regions responsible for a large part of this variation. Currently, we perform fine mapping and validation of the candidate loci. Cytidine analog zebularine-induces DNA damage in plants. Cytidine analogs, zebularine or 5-aza-cytidine, are used in epigenetic research as suppressors of gene silencing. However, their molecular effects are only incompletely understood. We found that zebularine treatment triggers DNA damage response that is signaled additively by ATR and ATM kinases and is repaired preferentially by synthesis-dependent strand annealing-type of homologous recombination. Our current efforts are focused on understanding the mechanisms of DNA damage induction and repair by cytidine analogs in plants. Epigenetic control of transposon silencing under ambient and stress conditions and role of transposons in genome evolution Transposons occur in all known eukaryotic genomes. Although considered as excessive genomic fraction, there is growing evidence on their importance for specific biological processes (e.g. Lisch, 2013). However, spurious transposon activity may have deleterious effects on genome stability (Devos et al., 2002). Therefore, transposons are suppressed by transcriptional gene silencing (TGS), an epigenetic control mechanism that creates and maintains constitutively repressive chromatin state. Apical meristems are hotspots of epigenetic silencing. Recently, we have discovered a novel RNA directed DNA Methylation (RdDM) function that reinforces silencing of repetitive elements during early vegetative growth. In apical meristems, de novo DNA methylation activity of RdDM compensates for zebularine-reduced silencing and thus prevents repetitive element transcription. Loss of this activity allows inheritance of activated repeats in somatic tissues and partially also into the progeny. Increased transcription of many chromatin-related genes in apical meristems suggests that they function as vegetative epigenetic checkpoints. To find out more read our recent publication Full Text ONSEN – transposable element exploring host necessity for defense. Successful life cycle of a retrotransposon includes its amplification by the copy-paste mechanism. However, this is strongly opposed by epigenetic silencing. Recent studies have identified a heat-responsive COPIA-type retroelement named ONSEN that can transpose in the Pol IV defective background. This activation is enabled by the presence of several heat-responsive elements in its long terminal repeats. We use Arabidopsis lyrata and other related species in order to understand evolution of ONSEN heat-responsiveness in Brassicaceae. Transposons contribute to the emergence of new genes. Animal data suggest that LINE retrotransposons play an important role in gene duplication. Occasionally, retrotransposon reverse transcriptase processes a gene-derived mRNA and integrates it into the genome as an intron and promoter-less copy. Although such retrogenes have been found in plants, their biology and evolution are poorly understood. We identified 251 retrogenes in A. thaliana, corresponding to 1% of protein-coding genes. They are frequently transcribed in tissues other than their parental genes and many reach their transcription maximum in pollen, the tissue analogous to animal spermatocytes, where up-regulation of retrogenes has been found previously. However, the underlying mechanisms and function are currently unknown. See original publication Full text Bibliography Atwell, s., Huang, Y.S., Vilhjalmsson, B.J., Willems, G. et al. Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465:627-631 (2010). Berr, A., Pecinka, A., Meister, A., Kreth, G. et al. Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata. Plant Journal 48:771-783 (2006). Devos, K.M., Brown, J.K.M., Bennetzen, J.L.. Genome Size Reduction through Illegitimate Recombination Counteracts Genome Expansion in Arabidopsis. Genome Research 12:1075–1079 (2002). He, F., Zhang, X., Hu, J.Y., Turck, F. et al. (2012): Widespread interspecific divergence in cis-regulation of transposable elements in the Arabidopsis genus. Molecular Biology and Evolution 29:1081-1091 (2012). Hollister, J.D., Smith, L.M., Guoc, Y.-L., Ott, F. et al. Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata. PNAS 108:2322-2327 (2011). Hu T.T., Pattyn, P., Bakker, E.G., Cao, J. et al. The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nature Genetics 43:476–481 (2011). Kakutani, T., Jedelloh, J.A., Flower, S.K., Munakata, K. and Richards, E.J. Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proceedings of the National Academy of Sciences USA 93:12406-12411 (1996). Mandakova, T., Joly, S., Krzywinski, M., Mummenhoff, K. and Lysak, M.A. Fast Diploidization in Close Mesopolyploid Relatives of Arabidopsis. Plant Cell 22:2277-2290 (2010). Mathieu, O., Reinders, J., Čaikovski, M., Smathajitt, C. and Paszkowski, J. Transgenerational Stability of the Arabidopsis Epigenome Is Coordinated by CG Methylation. Cell 130:851-862 (2007). Rizzini L, Favory JJ, Cloix C, Faggionato D et al. Perception of UV-B by the Arabidopsis UVR8 protein. Science 332:103-106 (2011). Soppe W.J.J, Jacobsen S.E., Alonso-Blanco, C., Jackson, J.P. et al. The Late Flowering Phenotype of fwa Mutants Is Caused by Gain-of-Function Epigenetic Alleles of a Homeodomain Gene. Molecular Cell 6:791–802 (2000). Steimer, A., Amedeo, P., Afsar, K., Fransz, P., Mittelsten Scheid, O. and Paszkowski, J. Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis. Plant Cell 12:1165–1178 (2000). Sterken, R., Kiekens, R., Boruc, J., Zhang, F. et al. Combined linkage and association mapping reveals CYCD5;1 as a quantitative trait gene for endoreduplication in Arabidopsis. PNAS doi: 10.1073/pnas.1120811109 (2012). The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-815(2000).