Chromatin dynamics in pollen mother cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis

Abstract

Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMC) committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition toward the male reproductive lineage. Here we show that Arabidopsis PMC differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMC). This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages.

Keywords: Arabidopsis; chromatin; histone modifications; histone variants; pollen mother cells.

Figure 1

Nuclear reorganization in differentiating PMC. (A) Specification of PMC in the early Arabidopsis anther locule. A representative image of an anther locule stained for DNA is shown (Propidium iodide, PI, red), differential interference contrast, and the DIC image overlaid with PI counterstaining. The male sporangium forming the PMC is indicated by a white dotted line. Scale bar: 10 μm. Compared to surrounding somatic cells like tapetum nuclei, PMC nuclei are marked by significantly enlarged nuclear and nucleolar size, as is shown by the images on the right panel. Comparison of nuclear volume (B), heterochromatin content (C, RHF, relative heterochromatin fraction) and chromocenter number (D) between PMC (shown in white dotted line) and epidermal cells was based on quantitative analyses on 3D reconstructed whole-mount, embedded young anthers. PMC, white bars; Epidermal cells, gray bars. The number of nuclei analyzed was given in each bar (n). Differences between chromatin of PMC and epidermal cells in replicate quantitative measurements were assessed using a two-tailed Welch's t-test (^***P < 0.001). Error bars represent the standard deviation to the mean (s.e.m).

Figure 2

Dynamic nuclear distribution of GFP-tagged histone variants in developing anthers. (A,B) Expression of GFP-tagged linker histone variants H1.1 and H1.2 in the developing anther. GFP fluorescence was hardly detectable in differentiating PMC (images at the left and middle panels) and the tetrad (images at the right panel), which indicates an eviction of linker histone H1 in PMC and the tetrad. (C) Expression of the GFP-tagged histone variant H2A.Z in the developing anther. GFP-tagged H2A.Z is well expressed in surrounding somatic cells in the anthers, but absent in PMC (images at the left and middle panels). GFP-H2A.Z is restored after meiosis in the tetrad (the image at the right panel). PMC are marked by white dotted line. Green, GFP fluorescence.

Figure 3

Immunodetection and quantification of whole-mount anther locules reveal distinct patterns of chromatin modification in PMC. Global levels of H3K27me1 (A), H3K27me3 (B), H3K4me3 (C), and H3K4me2 (D) were measured in PMC nuclei (white dotted line) and surrounding somatic cells. The fluorescence intensity (FI) corresponding to immune-signals intensity (Ab) over propidium iodide intensity (PI) in PMC nuclei is expressed relative to the corresponding Ab/PI ratio in surrounding cells. The quantifications were done in 3-dimensional reconstructions of confocal images of whole-mount embedded anthers using Imaris (see Methods, She et al., 2013, 2014). Representative images are shown for the antibody (Ab, green), DNA (Propidium iodide, PI, red), and the Ab fluorescence image overlaid with PI counterstaining (Ab/PI). Scale bar: 10 μm. PMC, white bars; Surrounding somatic cells, gray bars. The number of nuclei analyzed was given in each bar (n). Differences between chromatin of PMC and surrounding somatic cells were assessed using a two-tailed Welch's t-test (^***P < 0.001). Error bars represent the standard deviation to the mean (s.e.m).