DNA damage response (DDR) is an essential mechanism by which living organisms maintain their genomic stability. In plants, DDR is important also for normal growth and yield. Here, we explored the DDR of a temperate model crop barley (Hordeum vulgare) at the phenotypic, physiological, and transcriptomic levels. By a series of in vitro DNA damage assays using the DNA strand break (DNA-SB) inducing agent zeocin, we showed reduced root growth and expansion of the differentiated zone to the root tip. Genome-wide transcriptional profiling of barley wild-type and plants mutated in DDR signaling kinase ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (hvatr.g) revealed zeocin-dependent, ATR-dependent, and zeocin-dependent/ATR-independent transcriptional responses. Transcriptional changes were scored also using the newly developed catalog of 421 barley DDR genes with the phylogenetically-resolved relationships of barley SUPRESSOR OF GAMMA 1 (SOG1) and SOG1-LIKE (SGL) genes. Zeocin caused up-regulation of specific DDR factors and down-regulation of cell cycle and histone genes, mostly in an ATR-independent manner. The ATR dependency was obvious for some factors associated with DDR during DNA replication and for many genes without an obvious connection to DDR. This provided molecular insight into the response to DNA-SB induction in the large and complex barley genome.

• MeSH
• ATM protein genetika   metabolismus   MeSH
• bleomycin * MeSH
• DNA MeSH
• ječmen (rod) * genetika   metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ATM protein MeSH
• bleomycin * MeSH
• DNA MeSH
• Zeocin MeSH Prohlížeč

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

• Klíčová slova
• 3D organization, Nucleus, RNA Pol II, SIM, STED imaging, chromatin, chromosome, crossovers, image analysis, meiosis, metaphase, mitosis, nuclear bodies, nuclear speckles, oligo FISH, pachytene, quantification, segmentation, spatial distribution, transcription factories,
• MeSH
• buněčné jádro * MeSH
• chromatin * MeSH
• fluorescenční mikroskopie MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• průběh práce MeSH
• zelené fluorescenční proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin * MeSH
• zelené fluorescenční proteiny MeSH

Wild barley is abundant, occupying large diversity of sites, ranging from the northern mesic Mediterranean meadows to the southern xeric deserts in Israel. This is also reflected in its wide phenotypic heterogeneity. We investigated the dynamics of DNA content changes in seed tissues in ten wild barley accessions that originated from an environmental gradient in Israel. The flow cytometric measurements were done from the time shortly after pollination up to the dry seeds. We show variation in mitotic cell cycle and endoreduplication dynamics in both diploid seed tissues (represented by seed maternal tissues and embryo) and in the triploid endosperm. We found that wild barley accessions collected at harsher xeric environmental conditions produce higher proportion of endoreduplicated nuclei in endosperm tissues. Also, a comparison of wild and cultivated barley strains revealed a higher endopolyploidy level in the endosperm of wild barley, that is accompanied by temporal changes in the timing of the major developmental phases. In summary, we present a new direction of research focusing on connecting spatiotemporal patterns of endoreduplication in barley seeds and possibly buffering for stress conditions.

• Klíčová slova
• Endoreduplication, Hordeum vulgare ubsp. spontaneum, endosperm, seed development, super cycle value,
• MeSH
• DNA rostlinná genetika   MeSH
• endosperm genetika   MeSH
• genetická variace genetika   MeSH
• ječmen (rod) genetika   MeSH
• polyploidie MeSH
• populační genetika metody   MeSH
• semena rostlinná genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Izrael MeSH
• Názvy látek
• DNA rostlinná MeSH