Publications des scientifiques de l'IRD

She W. J., Grimanelli Daniel, Baroux C. (2014). Anefficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in Arabidopsis. Jove-Journal of Visualized Experiments, (88), p. e51530 [9 p.]. ISSN 1940-087X.

Titre du document
Anefficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in Arabidopsis
Année de publication
2014
Type de document
Article référencé dans le Web of Science WOS:000349290100048
Auteurs
She W. J., Grimanelli Daniel, Baroux C.
Source
Jove-Journal of Visualized Experiments, 2014, (88), p. e51530 [9 p.] ISSN 1940-087X
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.
Plan de classement
Sciences fondamentales / Techniques d'analyse et de recherche [020] ; Sciences du monde végétal [076]
Localisation
Fonds IRD [F B010063952]
Identifiant IRD
fdi:010063952
Contact