Chromatin organization and its interactions are essential for biological processes, such as DNA repair, transcription, and DNA replication. Detailed cytogenetics data on chromatin conformation, and the arrangement and mutual positioning of chromosome territories in interphase nuclei are still widely missing in plants. In this study, level of chromatin condensation in interphase nuclei of rice (Oryza sativa) and the distribution of chromosome territories (CTs) were analyzed. Super-resolution, stimulated emission depletion (STED) microscopy showed different levels of chromatin condensation in leaf and root interphase nuclei. 3D immuno-FISH experiments with painting probes specific to chromosomes 9 and 2 were conducted to investigate their spatial distribution in root and leaf nuclei. Six different configurations of chromosome territories, including their complete association, weak association, and complete separation, were observed in root meristematic nuclei, and four configurations were observed in leaf nuclei. The volume of CTs and frequency of their association varied between the tissue types. The frequency of association of CTs specific to chromosome 9, containing NOR region, is also affected by the activity of the 45S rDNA locus. Our data suggested that the arrangement of chromosomes in the nucleus is connected with the position and the size of the nucleolus.

• Klíčová slova
• 3D immuno-FISH, chromosome painting, chromosome territory, microscopy, rice, spatial organization,
• Publikační typ
• časopisecké články MeSH

Visualization of chromosome territories is a challenging task in plant genomes due to the lack of chromosome-specific probes, especially in species with large genomes. On the other hand, combination of flow sorting, genomic in situ hybridization (GISH), confocal microscopy, and employment of software for 3D modeling enables to visualize and characterize chromosome territories (CT) in interspecific hybrids. Here, we describe the protocol for the analysis of CTs in wheat-rye and wheat-barley hybrids, including amphiploids and introgression forms, where a pair of chromosomes or chromosome arms from one species is introgressed into the genome of another species. In this way, the architecture and dynamics of CTs in various tissues and different stages of cell cycle can be analyzed.

• Klíčová slova
• 3D FISH, Chromosome territory, Confocal microscopy, Imaris, Interphase nucleus, Plant hybrid,
• MeSH
• buněčné jádro genetika   MeSH
• chromozomy rostlin * genetika   MeSH
• genom rostlinný MeSH
• hybridizace in situ MeSH
• zavlečené druhy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

• Klíčová slova
• auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
• MeSH
• Arabidopsis účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné jádro účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné kultury MeSH
• centrifugace metody   MeSH
• frakcionace buněk přístrojové vybavení   metody   MeSH
• hmotnostní spektrometrie MeSH
• homeostáza fyziologie   MeSH
• indoly metabolismus   farmakologie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• protoplasty chemie   MeSH
• průtoková cytometrie MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné buňky účinky léků   metabolismus   ultrastruktura   MeSH
• tabák účinky léků   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indole MeSH Prohlížeč
• indoly MeSH
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH

The ever-increasing interest in epigenetics comes from the fact that in the diverse life situations of organisms, e [...].

• MeSH
• chromatin metabolismus   MeSH
• epigeneze genetická * MeSH
• metylace DNA MeSH
• posttranslační úpravy proteinů MeSH
• rostliny metabolismus   MeSH
• Publikační typ
• úvodní články MeSH
• úvodníky MeSH
• Názvy látek
• chromatin MeSH