Genome-Wide Analysis Of Core Cell Cycle Genes And Function Identification Of Orysa; DEL1 In Rice

Cell proliferation is an important biological process. Progression of the eukarytotic cell cycle is primarily controlled by a highly conserved molecular machinery in which cyclin-dependent kinases play a central role. The core cell cycle regulators, such as cyclin-dependent kinases (CDK), cyclins, CDK inhibitors (CKIs), homologs of the retinoblastoma protein (Rb) and the E2F transcription factors (E2F), have been identified in eukaryotes. In higher plants, the cell cycle must be integrated into a complex system of histogenesis and organogenesis since the spatial and temporal regulation of cell division is essential to the development of plant form. The cell cycle is influenced by extracellular cues as well and plant hormones are important in the regulation of the cell cycle. Although a large number of core cell cycle genes have been identified in dicots, little is known about them in monocots. Rice is an important crop and model plant. To provide the basic information on the mechanism of cell cycle regulator in rice, it is important to identify the core cell cycle regulators and study their expression pattern in plant tissues.To identify core cell cycle genes in the rice genome, we conducted Blast searches against the rice protein database with query sequences of all previously published core cell cycle regulators from Arabidopsis. Total of 90 putative core cell cycle proteins were identified through the searches, and they belong to cyclin, CDK, E2F, CKI, CKS, Rb and Wee families, respectively. 44 cyclin proteins, 25 putative CDK proteins, 2 Kip/Cip proteins, 9 E2F transcription factors, 2 Rb-related proteins, 2 Wee proteins and 1 putative CKS were found in the rice genome. Of them, 41 were new or corrections of the existing annotations. The structures of the core cell regulators were relatively conserved between the rice and Arabidopsis genomes.To understand whether gene tandem or segmental duplications led to acquisition of new cell cycle, we mapped all cell cycle genes on the twelve different chromosomes. The results indicate that segmental duplication events had occurred in rice chromosomes and six chromosome segmental duplications events occurred were found on 9 of 12 chromosomes. Moreover, most of duplications were concentrated on chromosome 2,3,10 and 12. Segmental duplications lead to the formation of new gene. It is possible that one of gene was derived from another gene in the same group by segmental duplications of chromosome.Since gene expression pattern can provide important clues for gene function, we employed semi-quantitative RT-PCR to characterize the expression profiles of core cell cycle genes. The rice core cell cyele genes show diverse expression patterns. A majority of the cell cycle genes are expressed in most tissues tested, with various expression levels. In several type with multiple member, some members exhibit specific expression patterns. And, in many cases, the most closely related ones showed very similar patterns of expression, suggesting functional redundancy between the highly similar core cell cycle genes in rice and Arabidopsis.Further detailed studies of developmental expression, Orysa;CycA2;1, Orysa;CycB2;2, Orysa;CycU4;4 and Orysa;CDKG;1 were randomly selected for in situ hybridization analysis The in situ hybridization analysis supports the results of our RT-PCR analysis and suggests that the expression of core cell cycle genes was temporally and spatially regulated during rice plant development. Tissues and cell cycle-phase specific expression patterns will provide more clues for functional prediction of cell cycle genes.To determine which core cell cycle genes respond to cytokinin and auxin, the expression of the core cell cycle genes was examined for either upregulation or downregulation. We submerged roots of 10 day-old rice seedlings in a solution of IAA or 6-BA, and then harvested the seedlings at different time. After treatmenting with auxin, twenty-five genes were found to be upregulated and four genes were found to be downregulated. After cytokinin treatment, the transcript levels of 11 genes were increased, and by contrast, the expression of 26 genes was downregulated. The results suggesting that auxin and/or cytokinin may directly regulate the expression of the core cell cycle genes.To investigate the role of cell cycle gene during rice development, a putative Orysa;DEL1 was isolated which contains 441 amino acid residues. We conducted a comparison of Orysa;DEL1 proteins and phylogenetic analysis of the DEL genes in seed plants. The resultant tree revealed that the Orysa;DEL1 in rice consistent with trees obtained previously. The RRxYD motif which is a DNA recognition signature of E2F family have two copies in investigated Orysa;DEL1. Subsellular localization indicated that localize to the nucleus, although no obvious nuclear localization signal was predicted.To examine the spatial and developmental expression profile of Orysa;DEL1, semi-quantitative RT-PCR was performed with RNA from various organs/tissues. In vegetative organs, Orysa;DEL1 mRNA accumulated to its highest level in young leaves, and to relatively high levels in roots, and nodes. During seed development, transcript of Orysa;DEL1 was detected in young seeds and embryos. Compared to their expression in endosperm, Orysa;DEL1 was much more highly expressed in early seed and embryo. Further, the expression patterns of Orysa;DEL1 were analyzed using RNA in situ hybridization, and the signal was detected in root, young leaf and embryo.We addressed the function of Orysa;DEL1 using the RNA interference and overexpression strategies. The phenotype conferred by overexpression Orysa;DEL1 and correlated with downregulated Orysa;DEL1 mRNA in RNAi lines. Phenotypic analysis of transgenic plants revealed dramatic differences in trichomes development comparing to the corresponding controls. Scanning electron microscope analysis found that the macro hair were completely lost on transgenic leaves of overexpression Orysa;DEL1 transformants. The apical hairs were also lost on glume in transgenic line.In this study, we have identified and analyzed a large number of cell cycle genes from rice. Our results show that rice and Arabidopsis possess several large and complex familes of cell cycle genes. Although the diversification occurrec in some types or groups of the core cell cycle genes, the number of the core cell cycle genes is relatively conserved during evolution between the genomes of two species. Moreover, an orthologous relationship between genes from rice and Arabidopsis can form a basis for functional comparison using multiple approaches. Finally, Orysa;DEL1 function identification indicate that it plays an important role in trichomes formation or development of rice.