The fluid was then drawn through four layers of cheesecloth and strained and processed as for the leaf nuclei.
Interestingly, around the time of bolting, heterochromatin content of leaf nuclei decreases transiently.
In root cell nuclei isolated from the meristematic zone of 15 day old plants, the two NORs of A. thaliana origin are highly condensed and are colocalized with H3K9me2- enriched chromocenters (Figure 2B; see Tables S3 and S4 for quantitative data), as in leaf nuclei of mature plants (compare to Figure 1D).
Figure 1D (see also Tables S1 and S2) shows a comparison of cotyledon and mature leaf nuclei subjected to DNA fluorescence in situ hybridization (DNA-FISH) using an A. thaliana-specific rRNA gene probe (green signals) in conjunction with immunolocalization of H3K9me2 (red signals).
Leaf nuclei were prepared, as described by Jackson et al. [ 41].
We found that the average chromocenter number in crwn1, crwn2 and crwn3 leaf cell nuclei was similar to that seen in wild-type leaf cell nuclei and did not change dramatically as a function of nuclear size.
Taken together, these results show that in roots the A. thaliana rRNA genes that are subjected to nucleolar dominance are associated with H3 that is dimethylated on lysine 9 but are not associated with H3 that is trimethylated on lysine 4, in agreement with the ChIP dot-blot data (compare data of Figures 1 and 2) and previous studies using leaf cell nuclei of mature plants [14], [16].
The dashed line in Figure 4B depicts the expected nuclear size change in response to a reduction in endopolyploidy based on the established one-to-one relationship between nuclear volume (approximated by nuclear area in our measurements of isolated and flattened leaf cell nuclei, Additional file 6) and DNA content in wild type plants.
GFP alone was observed in the cytoplasm and nucleus of leaf epidermal cells in 35S::GFP transgenic plants (Figure 5D).
Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1.
