| dc.description.abstract | The cell cycle controls cell division and proliferation of all eukaryote cells and is important to development of all multi-cellular organisms. The cell cycle of sexually reproducing organisms is founded by the zygote and needs to be re-established in the founding generations of new organism. Without control and synchronization of the cell-cycle, gametes cannot be assured that the DNA complement of both gametes is activated and numerically correctly represented; thus, neither double fertilization nor embryo formation would be possible. According to studies in all eukaryotes, including yeast, animals and the model plant Arabidopsis, the cell cycle consists of four different phases (M, G1, S and G2), each of which is tightly regulated at checkpoints by the cooperation of several critical regulatory factors.
However, due to the difficulty in accessing to gametic and zygotic cells in flowering plants, which are controlled in single cells deeply embedded in multiple tissues, little is known about how molecular mechanisms underlying the initiation of plant embryogenesis may reflect or contrast from such systems in other eukaryotes. Fortunately, Dr. Russell lab has developed an approach of cell isolation that opened a pathway to examine the developmental status of rice gametes and zygotes, which make it possible to manipulate cell cycle control factors involved ultimately in the initiation of the seed formation.
In this project, we employed various approaches to identify the components involved in rice zygotic cell cycle and characterize their functions and regulation. The methods and results are reported in four chapters. The main points of each chapter will be stated as the following.
Firstly, we optimized the previous procedure to isolate more, pure and viable rice egg cells and zygotes from different developmental stages, and also developed a new efficient approach, Blender method, to purify rice sperm cells. Both isolated rice egg cells and sperm cells were identified with marker genes and used in the published study of siRNAs in rice gametes and zygotes. Our practice shows that the isolated rice gametes and zygotes are featured with viability, transparency, purity and intactness, and thus serve as an ideal system for research in cellular and molecular biology and biochemistry.
Secondly, based on our study, we established a model of arrested core complex involved in rice zygotic cell cycle control. It consists of four major regulatory components including CDKB1, CYCD5, KRP5 and KRP4. CDKB1 is the first major player in cell cycle progression; KRP4 and KRP5 function as CDKB1 inhibitors, as proved in the CDK activity analysis; more importantly, KRP5 and KRP4 act in a coordinate, or heterodimer-like, manner, as indicated in the results of Y2H, yeast growth in serial dilutions, BiFC and Kinase activity assay. Besides, this coordinate inhibition might exist in the cell cycle control of other living organisms.
Thirdly, as a novel rice F-box protein, Fb3 is preferentially expressed in rice egg cells and zygotes. It interacts with both KRP5 and KRP4 and mediates the degradation of these two KRP inhibitors through 26S proteasome pathway. This is evidenced in the protein degradation assay and supported by its reversal effect on KRP inhibition on the Kinase activity of CDKB. Our results identify Fb3 as a regulator of the two KRP inhibitors of rice zygotic cell cycle.
In addition, our phenotypic observation, genetic analysis, seed setting test and complementation trial in the rice mutant plants demonstrate that all KRP5, KR4 and Fb3 are rice specific proteins involved in initiation of rice seed formation. We also found that these mutations result in abnormal morphology in rice female germ units and compromised function in rice sperm cells.
Since rice genetics is well documented as the second sequenced flowering plant and the most abundant of human’s crops around the world, the knowledge from this effort may enrich our understanding of the molecular mechanism underlying cell cycle control in embryogenesis initiation; in agriculture, it may facilitate crop breeding for better rice production to meet the high food demand from increasing global population. | en_US |
