UC Davis

AbstractGenome-wide Redistribution of siRNAs in Rice Gametes and Zygotes

Gametes and zygote constitute a critical stage in the life cycle for all sexual organisms, including plants. For angiosperms, the seed is the basic reproductive unit, which is produced by double fertilization. The male gametophyte, pollen, contains two sperm cells. One of the sperm cells fuses with the egg cell to produce the zygote, which will develop into the embryo and the next sporophytic generation. The other sperm cell fuses with the diploid central cell to produce the endosperm, a nutritive tissue that supports the growth of the embryo or germinating seedling. Seeds — either the endosperm (e.g., rice, maize, wheat, and other cereals) or the embryo (e.g., soybean, peanut, and other pulses) — directly or indirectly account for most of the calorie human consume, and seeds are the product of double fertilization. Thus, understanding the biology of gametes and zygote has broad applications in biotechnology and agriculture. In this dissertation I detail an aspect of the biology of rice gametes and zygote: the small RNA transcriptome and its implications regarding epigenome and plant development. In the first chapter I introduce 1) zygotic genome activation in plant, a process during which the fertilized egg cell transition from the gametic cell fate to the embryonic and totipotent cell fate; and 2) epigenomic reprogramming that was predicted or reported before and after fertilization in angiosperms. In the second chapter, I present detail protocols for isolating rice gametes, which were used to generate data presented in later chapters of this dissertation. In the third chapter, I present results of a study characterizing the small RNA transcriptomes and DNA methylomes of rice gametes. The results indicate that a genome-wide redistribution of 24-nt siRNAs has occurred in rice gametes, which was unexpected and not predictable from our canonical understanding of siRNA functions. However, in both gametes, the patterns of CHH methylation, a strong indicator of RNA-directed DNA methylation, remain similar to each other and to vegetative tissues. These findings are suggestive to unexplored roles of gamete small RNAs. In the fourth chapter, I present results of a study characterizing the small RNA transcriptome of rice zygotes. The results indicate that wide-spread redistribution of siRNAs occurred in zygote, and newly detected siRNA loci in zygote have a distribution similar to that of canonical siRNA loci detected in embryo and seedling. In addition, zygote siRNA loci, but not egg siRNA loci, were associated with hypermethylation in mature embryo. These findings suggest that resetting of the gametic epigenome towards the canonical vegetative pattern is initiated in the zygote, setting the stage for RdDM activity during embryogenesis before the first embryonic division. Lastly, I briefly summarize key findings of my dissertation and discuss possible future experiments in the final chapter of this dissertation.