An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in <em>Arabidopsis</em>

Abstract

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues. Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

Probing the 3D architecture of the plant nucleus with microscopy approaches: challenges and solutions

“…Figure 2 Whole-mount immunostaining also proves powerful for analysing and quantifying chromatin modifications and distribution patterns in specific cell types. Examples include chromatin studies in the gametes of ovules and anthers and in developing embryos in different species:-Arabidopsis, rice and maize [89][90][91][92]. Figure 2(b) shows an example of high-resolution chromatin imaging in wholemount fixed ovules [89,91] showing immunostaining of H3K27me1, a heterochromatin-specific histone modification (Figure 2 Empowered by Imaris (Bitplane AG, CH).…”

“…b1 shows a max.projection (inset: overlay with the transmission DIC channel) and detail of the nucleus of the spore mother cell after 3D segmentation (yellow insets) as max.projection and with orthogonal slice views. This image quality allows for signal quantification and measurements of relative histone modification levels [91]. b2 shows a mature embryo sac before fusion of the polar nuclei (pn).…”

“…Another motivation for deploying approaches using isolated nuclei is the challenge of applying the fluorescence probe (dye, antibody or RNA/DNA hybridization probe) homogenously in a whole mount sample. Although helpful protocols exist [86][87][88]91] among others), scientists can be defeated by their complexity and lack of robustness. In this situation, it might be necessary to compromise on preserving tissue integrity by isolating nuclei or cells.…”

“…This includes data normalisation, verification of the data distribution and evaluation of the variance. Examples of normalisation include expressing the fluorescence intensity of a given antibody relative to that of another, co-localising antibody or dye [91], and the distance between objects relative to the nuclear size/diameter. The type of data (discrete vs continuous) and the shape of the distribution (e.g.…”

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation

“…The protocol was adapted from She et al (2014). Roots of 5-d-old plants were fixed in fixation buffer (13 PBS, 2 mM EGTA, 1% formaldehyde, 10% DMSO, and 1% Tween 20) for 30 min at room temperature and then mounted in 5% acrylamide on a microscope slide.…”