title: Optimized Methods for the Isolation ofArabidopsisFemale Central Cells and Their Nuclei.
abstract: TheArabidopsisfemale gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations ofArabidopsiscentral cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights intoArabidopsisreproduction.
authors: Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
authors: Kyunghyuk
authors: K
authors: Park
authors: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors: Jennifer M
authors: JM
authors: Frost
authors: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors: Adam James
authors: AJ
authors: Adair
authors: Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
authors: Dong Min
authors: DM
authors: Kim
authors: Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
authors: Hyein
authors: H
authors: Yun
authors: Department of Science, Seoul Foreign School, Seoul 09723, Korea.
authors: Janie S
authors: JS
authors: Brooks
authors: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors: Robert L
authors: RL
authors: Fischer
authors: Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
authors: Yeonhee
authors: Y
authors: Choi
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics; D002467:Cell Nucleus / Q000502:physiology; D005718:Gametogenesis; D030821:Plants, Genetically Modified / Q000166:cytology
keywords: Arabidopsis central cell; embryo sac; nuclei isolation; protoplast