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Transcriptomics of Arabidopsis sperm cells at single-cell resolution

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Abstract

Key message

We present a detailed protocol for isolation of single sperm cells and transcriptome analysis to study variation in gene expression between sperm cells.

Abstract

Male gametophyte development in flowering plants begins with a microspore mother cell, which upon two consecutive cell divisions forms a mature pollen grain containing a vegetative nucleus and two sperm cells. Pollen development is a highly dynamic process, involving changes at both the transcriptome and epigenome levels of vegetative nuclei and the pair of sperm cells that have their own cytoplasm and nucleus. While the overall transcriptome of Arabidopsis pollen development is well documented, studies at single-cell level, in particular of sperm cells, are still lacking. Such studies would be essential to understand whether and how the two sperm cells are transcriptionally different, in particular once the pollen tube grows through the transmitting tissue of the pistil. Here we describe a detailed protocol for isolation of single sperm cells from growing pollen tubes and analysis of their transcriptome.

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Acknowledgements

The authors would like to acknowledge the Genomics Unit and Bioinformatics Unit of IGC. The Genomics Unit was partially supported by ONEIDA Project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI—”Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa 2020″ and by national funds from FCT—”Fundação para a Ciência e a Tecnologia”. This work was funded by FCT ERA-CAPS project EVOREPRO (ERA-CAPS-0001-2014). CSM acknowledges a doctoral fellowship from FCT (PD/BD/114362/2016) under the Plants for Life PhD Program. JDB received salary support from FCT through an “Investigador FCT” position.

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Authors and Affiliations

  1. Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal

    Chandra Shekhar Misra, Mário R. Santos, Mariana Rafael-Fernandes, Nuno P. Martins, Marta Monteiro & Jörg D. Becker

  2. Instituto de Tecnologia Química e Biológica, Avenida da República, 2780-157, Oeiras, Portugal

    Chandra Shekhar Misra

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  1. Chandra Shekhar Misra
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Correspondence to Jörg D. Becker.

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A contribution to the special issue ‘Cellular Omics Methods in Plant Reproduction Research’.

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Misra, C.S., Santos, M.R., Rafael-Fernandes, M. et al. Transcriptomics of Arabidopsis sperm cells at single-cell resolution. Plant Reprod 32, 29–38 (2019). https://doi.org/10.1007/s00497-018-00355-4

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