H3.1 Eviction Marks Female Germline Precursors in Arabidopsis

Abstract

In flowering plants, germline precursors are differentiated from somatic cells. The female germline precursor of Arabidopsis thaliana is located in the internal (nucellar) tissue of the ovule, and is known as the Megaspore Mother Cell (MMC). MMC differentiation in Arabidopsis occurs when a cell in the subepidermal layer of the nucellar apex enters the meiotic program. Increasing evidence has demonstrated that MMC specification is a plastic process where the number and developmental outcome of MMCs are variable. During its differentiation, the MMC displays specific chromatin hallmarks that distinguish it from other cells within the primordium. To date, these signatures have been only analyzed at developmental stages where the MMC is morphologically conspicuous, and their role in reproductive fate acquisition remains to be elucidated. Here, we show that the histone 3 variant H3.1 HISTONE THREE RELATED 13 (HTR13) can be evicted in multiple subepidermal cells of the nucellus, but that H3.1 eviction persists only in the MMC. This pattern is established very early in ovule development and is reminiscent of the specific eviction of H3.1 that marks cell cycle exit in other somatic cell types, such as the root quiescent center (QC) of Arabidopsis. Our findings suggest that cell cycle progression in the subepidermal region of the ovule apex is modified very early in development and is associated with plasticity of reproductive fate acquisition.

Keywords: Arabidopsis; Cell cycle; histone H3; ovule development; plasticity; reproductive competence.

Figure 1

Specific dynamics of HTR13-GFP (H3.1) chromatin-deposition during early Arabidopsis ovule development. (a–c) Representative image of cleared ovules preceding stage 1-I (stages 0-I to 0-III), used for quantitative analysis. (d–f) Representative images of emerging ovule primordia showing the three different HTR13 patterns detected in subepidermal cells of the nucellus: (d): Ovule primordium showing no eviction of HTR13 at the MMC (Megaspore Mother Cell). Green arrow in the inset shows the nuclei of the MMC with GFP signal. (e) Ovule primordium showing HTR13 eviction (white arrow) in a single subepidermal cell. (f) Ovule primordium showing HTR13 eviction in two subepidermal cells (white arrows). (g) Frequency of ovule primordia exhibiting the distinct HTR13 patterns within subepidermal cells in the nucellus. Standard Error (SE) is indicated, n = number of ovules observed. Two-tailed Fischer’s test was used to compare the proportion of eviction in a single cell between stage 1-I and 2-II. *** p < 0.001. (h–i) 3D reconstructions of cell surfaces showing nuclei GFP signal detection (red stars) in all epidermal cells (grey surfaces), and only in one out of three (h) or in one out of two (i) subepidermal cells (yellow surfaces). (j) Ovule at stage 2-I showing eviction in the MMC and not in companion cells. (k) Ovule at stage 2-II showing eviction in two enlarged subepidermal cells. (l) 3D reconstruction of cell surfaces showing nuclear GFP signal in all epidermal cells, in MMC neighbor cells (magenta surfaces, n.c.) and eviction in the MMC (yellow surface). (m) Working model: At early stages of ovule primordium development, HTR13 eviction and cell cycle exit occur in several subepidermal cells, distinguishing them from the somatic tissues (soma). This eviction persists in the MMC, which will undergo meiosis. Conversely, HTR13 is gradually re-incorporated in the neighbor cells of the MMC, which re-enter the soma. Squares mark inset regions. Dashed lines mark MMC or candidate MMCs. Green: GFP fluorescence; Magenta: cell wall marker Renaissance SR2200; scale bars: 10 μm. (a–c) DIC microscopy. (d–f,j,k) Laser Scanning Confocal Microscopy. (h,i,l) 3D reconstruction of cell surfaces after epidermal and subepidermal cell segmentation and automated nuclei detection.