PMID- 24998753
OWN - NLM
STAT- MEDLINE
DCOM- 20141219
LR  - 20240321
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 88
DP  - 2014 Jun 19
TI  - An efficient method for quantitative, single-cell analysis of chromatin 
      modification and nuclear architecture in whole-mount ovules in Arabidopsis.
PG  - e51530
LID - 10.3791/51530 [doi]
LID - 51530
AB  - 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.
FAU - She, Wenjing
AU  - She W
AD  - Institute of Plant Biology and Zurich-Basel Plant Science Center, University of 
      Zurich.
FAU - Grimanelli, Daniel
AU  - Grimanelli D
AD  - Institut de Recherche pour le Developpement (UMR 232), Centre National de la 
      Recherche Scientifique (ERL 5300), Universite de Montpellier II.
FAU - Baroux, Celia
AU  - Baroux C
AD  - Institute of Plant Biology and Zurich-Basel Plant Science Center, University of 
      Zurich; cbaroux@botinst.uzh.ch.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20140619
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (Chromatin)
RN  - 0 (DNA, Plant)
SB  - IM
MH  - Arabidopsis/*chemistry/metabolism/*ultrastructure
MH  - Cell Nucleus/chemistry/ultrastructure
MH  - Chromatin/*chemistry/metabolism
MH  - DNA, Plant/analysis
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Ovule/*chemistry/*ultrastructure
MH  - Single-Cell Analysis/*methods
PMC - PMC4195603
EDAT- 2014/07/08 06:00
MHDA- 2014/12/20 06:00
PMCR- 2016/06/19
CRDT- 2014/07/08 06:00
PHST- 2014/07/08 06:00 [entrez]
PHST- 2014/07/08 06:00 [pubmed]
PHST- 2014/12/20 06:00 [medline]
PHST- 2016/06/19 00:00 [pmc-release]
AID - 51530 [pii]
AID - 10.3791/51530 [doi]
PST - epublish
SO  - J Vis Exp. 2014 Jun 19;(88):e51530. doi: 10.3791/51530.