Abstract
Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.
Highlights
Materials and methodsThe Arabidopsis thaliana accessions Col (N1092), Landsberg erecta (Ler) (NW20), Wassilewskija (Ws) (Ws-2), and Cvi were obtained from the Arabidopsis Biological Resource Stock Center (ABRC, Nottingham, UK)
To facilitate quantification of nuclear and chromatin morphometry, we introduced the term relative heterochromatin fraction (RHF), which represents the portion of total fluorescence intensity of all chromocenters relative to the fluorescence intensity of the entire nucleus
We focus on parameters that are related to the distribution of genomic DNA and heterochromatic sequences and include size, DNA content, DNA density, RHF, and number of chromocenters (CCs)
Summary
The Arabidopsis thaliana accessions Col (N1092), Landsberg erecta (Ler) (NW20), Wassilewskija (Ws) (Ws-2), and Cvi were obtained from the Arabidopsis Biological Resource Stock Center (ABRC, Nottingham, UK). Post-hybridization washes were performed in 50% formamide, 2 × SSC (pH 7.0) for 3 × 5 min at 42 °C, followed by 2 × SSC at room temperature for 3 × 5 min, dehydration through an ethanol series and staining with 2 μg/mL DAPI on slides. In order to semi-automatically analyze high numbers of raw (unprocessed) images, we applied our in-house developed macro in ImagePro-Plus (Media Cybernetics, Silver Spring, MD, USA) for the morphometric analysis to segment the nuclei and measure size (number of pixels) and fluorescence intensity of the interphase nucleus and individual chromocenters by a threshold algorithm (Pavlova et al 2010). The fraction of TEs in CCs is calculated as follows: TE fraction in CC = RHF × Genome size − total amount of 45S and 180 bp DNA total amount of TE. The fraction of TEs in euchromatin is determined by subtracting the fraction TEs in CC from 1
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