SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content

Schoft, Vera K.; Chumak, Nina; Bindics, János; Ślusarz, Lucyna; Twell, David; Köhler, Claudia; Tamaru, Hisashi

doi:10.1007/s00497-015-0258-2

Cited by 18 publications

(22 citation statements)

References 32 publications

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“…Pollen nuclei were isolated as previously described 47 , 49 . Approximately 50.000 nuclei were purified by FACS and used to construct sequencing libraries of bisulfite-treated DNA using the Pico Methyl-Seq kit (Zymo Research), according to manufacturer instructions.…”

Section: Methodsmentioning

confidence: 99%

Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis

et al. 2018

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Chromosome dosage plays a significant role in reproductive isolation and speciation in both plants and animals, but underlying mechanisms are largely obscure1. Transposable elements can promote hybridity through maternal small RNA2, and have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here, we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of LTR-retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21 to 22-nucleotide small RNA in a dose dependent fashion via RNA polymerase IV. We show that these epigenetically activated small-interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (“the triploid block”), and that natural variation for miR845 may account for “endosperm balance” allowing formation of triploid seeds. Targeting the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.

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Section: Methodsmentioning

confidence: 99%

Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis

et al. 2018

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“…These approaches would allow the binding between transcription factors and their target loci to be resolved, and to decipher how these interactions are integrated with the changing chromatin landscape. The precision of such methods will be further enhanced by the use of fluorescence-activated cell sorting (FACS) and single cell transcriptome analysis Santos et al, 2017;Schoft et al, 2015). Future studies using established and newly emerging models promise major advances in our understanding of the development and evolution of male gametogenesis in plants.…”

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confidence: 99%

The evolution and patterning of male gametophyte development

Hackenberg

Twell

2019

Current Topics in Developmental Biology

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1. The evolutionary history of the male gametophyte 1.1. The origins of spermatogenesis in green plants 1.2. The sporophyte was key for terrestrial plant evolution 1.3. Heterospory and the origin of microgametophytes 1.4. Adaptations of seed plant microgametophytes 2. Male germline development in angiosperms 2.1. Asymmetric division and cell fate determination 2.2. Cell cycle control and male gametophyte patterning 2.3. Transcriptional control of male gametophyte patterning 2.4. Chromatin remodelling in male gametophyte development 2.5. Communication between non-germ and germ cells 3. Molecular evolution of the male germline 3.1. Motile plant and animal sperm share common features 3.2. Gene expression in male germ cells 3.3. The evolution of transcription factors regulating male gametogenesis 4. Perspective 5. Acknowledgements 2 6. References Abstract: The reproductive adaptations of land plants have played a key role in their terrestrial colonization and radiation. These encompass mechanisms used for the production, dispersal and union of gametes to support sexual reproduction. The production of small motile male gametes and larger immotile female gametes (oogamy) in specialized multicellular gametangia evolved in the charophyte algae, the closest extant relatives of land plants.Reliance on water and motile male gametes for sexual reproduction was retained by bryophytes and basal vascular plants, but was overcome in seed plants by the dispersal of pollen and the guided delivery of non-motile sperm to the female gametes. Here we discuss the evolutionary history of male gametogenesis in streptophytes (green plants) and the underlying developmental biology, including recent advances in bryophyte and angiosperm models. We conclude with a perspective on research trends that promise to deliver a deeper understanding of the evolutionary and developmental mechanisms of male gametogenesis in plants.

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“…DNA extraction was performed as described in Schoft et al (2011). RNA extraction was done according to Schoft et al (2015). The samples can also be fixed (Schoft et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

“…RNA extraction was done according to Schoft et al (2015). The samples can also be fixed (Schoft et al, 2015). Although we never tried, the samples should, in principle, survive flash freezing and storage at -80 °C .…”

Section: Methodsmentioning

confidence: 99%

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Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS

2015

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One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 10 6 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.

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SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content

Cited by 18 publications

References 32 publications

Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis

Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis

The evolution and patterning of male gametophyte development

Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS

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