2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions

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.

Keywords: Arabidopsis; Chromocenter; Heterochromatin; Nuclear phenotype; Quantitative analysis.

Fig. 1

Morphometric analysis of isolated nuclei. A Whisker-box plots of RHF (Y-axis) from three independent sets of DAPI-stained Ler nuclei (set 1, blue, n = 32; set 2, red, n = 31; set 3, green, n = 34), each of them analyzed with CHIAS, Image Pro + and Object Image. B Representative examples of nuclei (upper row) and whisker-box plots of RHF (Y-axis) from parenchyma/pavement nuclei (lower row), stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), Sytox-Green (green), and propidium iodide (PI; red). Boxes indicate boundaries of second and third quartiles of data distributions. Black bars within the boxes indicate the median, and the error bars (whiskers) indicate the Q1 and Q4 values within 1.5 times the interquartile range. Observations outside 1.5 times the interquartile range are indicated as dots. Violin plots designate phenotype distributions. Letters (A, B, C) and colors indicate statistical differences between groups, with different letters indicating significantly different groups (p < 0.05) per panel.

Fig. 2

Identification of nuclei from different cell types of a young Arabidopsis leaf. On the left is a schematic drawing of a cross section obtained from a cross-sectioned leaf drawing (https://commons.wikimedia.org/wiki/File:Cross_section_of_Arabidopsis_thaliana,_a_C3_plant..jpg). On the right are images of H2B-YFP-stained nuclei derived from optical confocal sections of different cell types and selected magnifications. The right column represents a magnification of the boxed areas (white dashed lines) displayed in the left column. From top to bottom: pavement cell nucleus (PC1), parenchyma nucleus (PC2), endopolyploid cell nucleus, vascular cell nucleus (VC), and guard cell nucleus (GC). Depth of the z-stack is 16 μm. Bar for scale represents 5 μm.

Fig. 3

Morphometric profiling of nuclei from different cell types. Whisker-box plots showing morphometric differences between cell types A and between organs C. B Principal component analysis on scaled parameters in different cell types reveals four clusters of accessions, with the GC cluster more separate from the other three. Boxes indicate the boundaries of the second and third quartiles of the data distribution. Black bars within the boxes indicate the median, and the error bars (whiskers) indicate Q1 and Q4 values within 1.5 times the interquartile range. Observations outside 1.5 times the interquartile range are indicated as dots. Violin plots designate phenotype distributions. Significance levels are indicated as letters above the bars and represent a two-side t-test assuming unequal variances, with different letters indicating significantly different groups (p < 0.05) per panel. Colors of the boxes are shared if statistically similar. Units on the Y-axis are arbitrary.

Fig. 4

Pearson correlation between parameters in different cell types. A Heatmap and B correlation matrix showing the pairwise correlation coefficients A and significance of correlations B between and among nuclear and chromocenter parameters. Color intensity indicates the strength of the correlation A and significance levels B. Positive correlations are indicated in red, negative correlations, in blue (see legend). Ranking is according to PC values. Cc, chromocenter; TE, transposon element; GC, guard cell; PC, pavement cell; VC, vascular cell; EC, endopolyploid cells. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5

Morphometric profiling of PC nuclei from different accessions. A Whisker-box plots showing morphometric differences between accessions. Boxes indicate the boundaries of the second and third quartiles of the data distribution. Black bars within the boxes indicate the median, and the error bars (whiskers) showing the values in Q1 and Q4 within 1.5 times the interquartile range. Observations outside 1.5 times the interquartile range are indicated as dots. Violin plots designate phenotype distributions. Significance levels are indicated as letters above the bars and represent a two-side t-test assuming unequal variances. Different letters indicate significant differences (p < 0.01) per panel. Colors of the boxes are shared if statistically similar. Units on the Y-axis are arbitrary. B Principal component analysis on scaled parameters in different accessions reveals two clusters of accessions.

Fig. 6

Pearson correlation between parameters in different accessions. A Heatmap and B correlation matrix, showing the pairwise correlation coefficient between and among nuclear and chromocenter parameters of the five tested accessions. Color intensity indicates the strength of the correlation A and significance levels B. Positive correlations are indicated in red, negative correlations, in blue (see legend). Ranking is according to PC values. Cc, chromocenter; TE, transposon element; GC, guard cell; PC, pavement cell; VC, vascular cell; EC, endopolyploid cells. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

Quantification of NOR-chromocenter association in Col, Cvi, C24, Ler, and Ws. The low percentages of detected NOR-CCs in Cvi and Ws indicates a high association of NOR-CCs. Lower panel shows representative examples of the 45S rDNA FISH signals in PC nuclei of the four accessions.