Pollen epigenetics

Biology learned in school and as a first year undergraduate is easily forgotten if it is not relevant to your current research. Today’s highlighted article required me to refresh my memory of plant germ line development, so I included my basic research here.

Highlighted article: Joseph P. Calarco, Filipe Borges, Mark T.A. Donoghue, Frédéric Van Ex, Pauline E. Jullien, Telma Lopes, Rui Gardner, Frédéric Berger, José A. Feijó, Jörg D. Becker and Robert A. Martienssen (2012) Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA. Cell 151:194-205.

Germline biosynthesis: A pollen mother cell undergoes meiosis to make haploid microspores, which unevenly split into a larger vegetative cell and a small generative cell. The generative cell splits symmetrically into two – these are the plant ‘sperm’ cells. Each pollen grain contains two sperm cells, which are surrounded by a vegetative cell. The vegetative nucleus contains completely decondensed heterochromatin, but DNA in generative nuclei is tightly condensed.

The female gametophyte develops from a megaspore mother cell. Both the megaspore mother cell and pollen mother cell are specified from somatic cells in developing flowers.

GFP staining in the two sperm nuclei and vegetative nucleus in the vegetative cell.

Bisulphite sequencing is a DNA sequencing method which determines methylation pattern by treating DNA with sodium bisulphite before sequencing it using a conventional DNA sequencing method. Bisulphite induces the conversion of unmethylated cytosines to uracil, but this is not a perfect technique so unmethylated DNA may be recorded as methylated. Additionally, bisulphite treatment can cause DNA degradation. Sequencing the DNA of interest multiple times, in the case of Calarco et al., anywhere from 7 to 17 times, improves reliability of the method. There is a brief overview of DNA methylation in this post.

Epigenetic inheritance is the transfer of imprinting, transposon silencing, or other epigenetic information, from one generation to the next. In animals, germline reprogramming removes most epigenetic information, but epigenetic inheritance is common in plants.

24 nt sRNA is a common class of siRNAs, and 24 nt sRNAs often guide RNA-directed DNA methylation. A recent review of RNA-directed DNA methylation is Haag and Pikaard, 2011.

Calarco et al. tracked the progress of DNA methylation throughout germline development by sequencing DNA methylation from pollen precursor, sperm, and vegetative cells. CHG methylation (where H = A, C or T) is more or less unchanged throughout the process, but CG methylation is lost in the vegetative nucleus. Schoft et al. (2011) had previously shown that DNA glycosylase DEMETER (DME), which is required for the demethylation of transposons, and its homologues are highly expressed in the vegetative pollen cell and somatic tissues but not in the sperm cell. Calarco et al. continued that work to identify DME targets, which do not have CG methylation in the vegetative nuclear DNA, suggesting that the loss of CG methylation is due to this set of DNA demethylases.

The greatest change in methylation profile is the loss of CHH in the conversion of a somatic cell to a microspore, and which is restored in the vegetative nucleus and in the embryo after fertilisation. This restoration is carried out by another DNA methyltransferase, which is guided by maternal 24nt sRNA.

This is the first time DNA methylation has been profiled throughout a germ cell ‘lifecycle’. Calarco et al. showed which type of genome imprinting can be inherited from parent plant to seedling, and how the process occurs.

Teaching resources: SAPS and the Nuffield Foundation both have activities for KS3 or 4 students to do with pollen and pollen tube growth.

Image credit: Shutterstock; Confocal image of pollen cell generated by Lynette Brownfield (University of Leicester), obtained via phys.org

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