Project Report

In sexually reproducing organisms, two copies of every non-sex linked genes are present: one copy is from mother and the other is from father. Generally both copies are expressed equally but a small subset of genes is differentially expressed, with a dominant expression from one parent. These maternally or paternally silenced genes are called imprinted genes. Imprinted genes in plants are suggested to influence seed size and quality. To date, imprinting is only found in embryo and endosperm of plants, the two main components of a seed. These tissues are formed through double fertilization. Pollen contains two sperms cells which fertilize the egg and the central cell, creating an embryo and endosperm, respectively. Genetic and molecular studies in plants have shown that presence or lost of DNA methylation regulates imprinted genes. DNA methylation is a well studied epigenetic mark that is associated with repression of genes and transposons without changing the underlying nucleotide sequences. Recently, we generated comprehensive single nucleotide maps of cytosine methylation and gene expression profiles in rice endosperm and embryo. In this study, we have identified a number of candidate imprinted genes. However, the mechanism by which these genes are selectively expressed in rice endosperm is still elusive. It has been suggested that imprinting is established in gametes and results in mono-allelic gene expression in endosperm and embryo after fertilization. Moreover, techniques to isolate gametes have been developed and optimized in many crop plants such as maize and rice. In order to unravel the question, I proposed to investigate DNA methylation in rice gametes. Through the EAPSI program, I have had an opportunity to collaborate with Dr. Takashi Okamoto, who is an expert in rice gamete isolation, in Tokyo Metropolitan University. The Okamoto lab has well established techniques for egg and sperm cell isolation. Upon arrival, I was trained to collect rice flowers of optimal stage for gamete isolation, to isolate egg and sperm cells, and to use an inverted microscope. I also optimized and modified the central cell isolation technique to collect more central cells with a shorter amount of time with relative ease. Over 200 eggs and sperm cells and over 50 central cells were collected manually during the EAPSI program. The genomic DNA from these cells will be treated with sodium bisulfite and sequenced to generate a map of DNA methylation at single-nucleotide resolution at UC Berkeley. The generated dataset will be analyzed to identify the mechanism of imprinting in rice seeds. Moreover, the genome-wide map of DNA methylation in gametes will be an important resource that can be used to understand the general landscape of DNA methylation in gametes in plants for the first time. Rice is an important crop plant that is the main nutrient source for much of the world. This project will identify the imprinted genes in rice endosperm and embryo and provide insights in the mechanisms of imprinting in crop plants. Studies have suggested that maternal and paternal genomes differentially influence the allocation of resources to seeds, which influence seed size and quality. Compared to seeds from parents with the same ploidy (in this case diploid), the seeds from a diploid mother and the tetrapolid father are larger while those from the reciprocal cross were smaller. This observation suggests that the paternal genome promotes greater resource investment in an individual developing seeds, while the maternal genome inhibits the allocation of resources. Here, the imprinted genes that are inhibited in maternal genome but expressed in paternal genome might be involved in producing larger seeds. The EAPSI program provided me a great opportunity to begin the project by collaborating with a Japanese host researcher and to generate a valuable resource in developing new technologies that increase crop yield and food production to feed a growing population and address the problem of hunger in our society.

National Science Foundation (NSF)
Office of International and Integrative Activities (IIA)
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Carter Kimsey
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Kim Min J
United States
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