Optimized Methods for the Isolation of ArabidopsisFemale Central Cells and Their Nuclei

Park, Kyunghyuk; Frost, Jennifer M.; Adair, Adam James; Kim, Dong-Min; Yun, Hyein; Brooks, Janie S.; Fischer, Robert L.; Choi, Yeonhee

doi:10.14348/molcells.2016.0209

Cited by 9 publications

(8 citation statements)

References 47 publications

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“…We used the INTACT (Isolation of Nuclei Tagged in specific Cell Types) approach (19,20) to isolate central cell nuclei from wild-type Arabidopsis plants. A reporter protein (Nuclear Targeting Fusion, NTF) consisting of a nuclear envelope localization domain fused to GFP was expressed from the DD7 central cell-specific promoter ( Fig.…”

Section: Resultsmentioning

confidence: 99%

DNA demethylation is initiated in the central cells of Arabidopsis and rice

Park

Kim

Vickers

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cytosine methylation is a DNA modification with important regulatory functions in eukaryotes. In flowering plants, sexual reproduction is accompanied by extensive DNA demethylation, which is required for proper gene expression in the endosperm, a nutritive extraembryonic seed tissue. Endosperm arises from a fusion of a sperm cell carried in the pollen and a female central cell. Endosperm DNA demethylation is observed specifically on the chromosomes inherited from the central cell in Arabidopsis thaliana, rice, and maize, and requires the DEMETER DNA demethylase in Arabidopsis. DEMETER is expressed in the central cell before fertilization, suggesting that endosperm demethylation patterns are inherited from the central cell. Down-regulation of the MET1 DNA methyltransferase has also been proposed to contribute to central cell demethylation. However, with the exception of three maize genes, central cell DNA methylation has not been directly measured, leaving the origin and mechanism of endosperm demethylation uncertain. Here, we report genome-wide analysis of DNA methylation in the central cells of Arabidopsis and rice-species that diverged 150 million years agoas well as in rice egg cells. We find that DNA demethylation in both species is initiated in central cells, which requires DEMETER in Arabidopsis. However, we do not observe a global reduction of CG methylation that would be indicative of lowered MET1 activity; on the contrary, CG methylation efficiency is elevated in female gametes compared with nonsexual tissues. Our results demonstrate that locus-specific, active DNA demethylation in the central cell is the origin of maternal chromosome hypomethylation in the endosperm.

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Section: Resultsmentioning

confidence: 99%

DNA demethylation is initiated in the central cells of Arabidopsis and rice

Park

Kim

Vickers

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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172

203

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“…To obtain tapetal cells, flower buds were harvested from the pA9::GFP and the pA9::GFP rdr2 lines and protoplasted as previously described 80 Supplementary Fig. 3a for the gate setting) were collected, examined under a fluorescence DM6000 microscope (Zeiss) for purity, and stored immediately at -80°C.…”

Section: Isolation Of Male Meiocytes Tapetal Cells and Spermmentioning

confidence: 99%

Nurse cell-derived small RNAs define paternal epigenetic inheritance inArabidopsis

Long

Walker

She

et al. 2021

Preprint

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The plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and facilitates meiosis. Why reprogramming is limited to the germline and how specific genes are chosen is unknown. Here, we demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by germline-specific siRNAs transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) via activity of the tapetum-specific chromatin remodeler CLASSY3. Remarkably, tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Finally, we demonstrate that these nurse cell-derived siRNAs (niRNAs) silence germline transposons, thereby safeguarding genome integrity. Our results reveal that tapetal niRNAs are sufficient to reconstitute germline methylation patterns and drive extensive, functional methylation reprogramming analogous to piRNA-mediated reprogramming in animal germlines.

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“…After 24 h of emasculation ( Park et al, 2016 ), fully matured stamens were picked from open flowers with tweezers. Pollen was rubbed onto the emasculated stigma for fertilization, under a dissection microscope.…”

Section: Methodsmentioning

confidence: 99%

“…This is, in part, due to difficulties in cell- and developmental stage-specific sampling resulting from the structural complexity and the small size of the Arabidopsis genome compared with that of animal model organisms, and difficulties in culturing cells. Furthermore, the isolation of specific cell-types often needs specialized protocols to obtain an appropriate amount of sample ( Deal and Henikoff, 2011 ; Ibarra et al, 2012 ; Park et al, 2016 ). Despite recent advances in the understanding of methylome changes in whole seeds ( Bouyer et al, 2017 ; Kawakatsu et al, 2017 ; Lin et al, 2017 ), methylation dynamics during embryo development in Arabidopsis are not fully understood.…”

Section: Introductionmentioning

confidence: 99%

An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo

Yoo

Park

Lee

et al. 2021

Molecules and Cells

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DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell-and tissuespecific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

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Optimized Methods for the Isolation of ArabidopsisFemale Central Cells and Their Nuclei

Cited by 9 publications

References 47 publications

DNA demethylation is initiated in the central cells of Arabidopsis and rice

DNA demethylation is initiated in the central cells of Arabidopsis and rice

Nurse cell-derived small RNAs define paternal epigenetic inheritance inArabidopsis

An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo

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