Genetic and epigenetic aspects of the plant male gametophyte

Heterochromatin is involved in multiple chromosomal processes such as silencing and recombinational suppression of repetitive DNA elements, chromosome segregation, and long-range chromatin interactions. In contrast to the rapidly growing number of factors implicated in heterochromatin formation and propagation, little is known about the mechanisms that actively reverse the process in any eukaryote. The purpose of the project is to understand the control and function of heterochromatin disassembly using the model plant Arabidopsis thaliana. Cytological and molecular studies of the vegetative and the sperm nuclei from Arabidopsis pollen have revealed a global heterochromatin disassembly process in the vegetative nucleus. Three complementary forward and reverse genetic approaches, for which the necessary tools are available, will be employed to isolate the genes involved in the process: (1) transgenic lines with fluorescent tags for the in vivo identification of the vegetative nucleus and for centromeric heterochromatin; (2) pre-selected T-DNA insertion lines carrying gametophytic mutations; and (3) tissue-specific transcriptome data. Additionally, possible genetic alterations, including DNA deletion and amplification, in the genomes of the vegetative and the sperm nuclei will be investigated using microarray-based technology. Results from these studies will allow novel insights into the plasticity and dynamics of heterochromatin.

Transposons are parasitic DNA elements that can move from place to place within the animal and plant genomes, unless inactivated by defense mechanisms. DNA methylation - a mark on genomic DNA made by the enzymatic addition of methyl group to cytosine bases - is associated with silencing of transposons. Reversibly, methylated DNA can be de-methylated by enzymes that excise and replace methyl-cytosines with cytosines. DNA de-methylation is associated with gene activation. DNA methyltransferase enzymes replicate pre-existing DNA methylation during cell divisions. However, the maintenance of methylation is not perfect, so a fraction of cytosines loses methylation during adult development from a fertilized egg. A critical problem in animal and plant reproduction is how transposons are stably silenced across generations. Our results suggest that active DNA de-methylation in plant companion cells reinforces de novo DNA methylation in gametes to re-establish full methylation and silencing of transposons for the next generation. Flowering plants form the female and male gametophytes, consisting of the gamete and its companion cell. The pollen vegetative cell (the companion cell of the sperm) forms a tube that transports two sperm cells to the ovule, where one fuses with the central cell (the companion cell of the egg) to form the placenta-like endosperm that nourishes the embryo, while the other fertilizes the egg to form the embryo. DNA glycosylase enzymes catalyze DNA de-methylation in plants. We found that the DEMETER DNA glycosylase of the flowering plant Arabidopsis thaliana de-methylates thousands of similar transposons in the genomes of the central and vegetative cells and induces de novo DNA methylation of complementary transposons in the egg and sperm cells. This new discovery is exciting because DEMETER functions to mediate two opposing processes, de-methylation and methylation of DNA, in two separate cells in the female and male gametophytes. What is the mechanism of this process? The de novo DNA methylation machine uses special types of RNA, so called small RNAs, to guide methylation of its complementary DNA sequences. Thus, the obvious deduction is that DNA de-methylation and transcriptional activation of transposons in gamete companion cells generates mobile signals - small RNAs - that move into the gametes to immunize against transposon activation over the plant sexual cycles. A dilemma of small RNA-directed DNA methylation is that cells need to transcribe transposon DNA into RNA and dice them to generate small RNAs. Transcription of transposons increases the risk of transposon activation and mobilization in the genome. Plant gamete companion cells provide a clever solution to this paradox: the companion central and vegetative cells, which are not transmitted to the progeny, risk their life to save the egg and sperm from being exposed to mobile transposons.