Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei
| Autor: | Dong Min Kim, Yeonhee Choi, Jennifer M. Frost, Janie S. Brooks, Kyunghyuk Park, Adam James Adair, Hyein Yun, Robert L. Fischer |
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| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine Genetics Gametophyte Gynoecium food and beverages Embryo Cell Biology General Medicine Biology biology.organism_classification 01 natural sciences Endosperm Cell biology Double fertilization 03 medical and health sciences Cell nucleus 030104 developmental biology medicine.anatomical_structure Arabidopsis medicine Ploidy Molecular Biology 010606 plant biology & botany |
| Zdroj: | Molecules and Cells. 39:768-775 |
| ISSN: | 0219-1032 1016-8478 |
| DOI: | 10.14348/molcells.2016.0209 |
| Popis: | The Arabidopsis female 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 of Arabidopsis central 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 into Arabidopsis reproduction. |
| Databáze: | OpenAIRE |
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