Genome-wide Analysis Of Core Cell Cycle Genes And Functional Determination Of Orysa; CycB1; 1 During Rice Seed Development

Cell division plays very important roles in many aspects of plant growth and development. Although extensive and systematic studies of core cell cycle genes such as cyclin-dependent kinases (CDK), cyclins, CDK inhibitors (CKIs), homologs of the retinoblastoma proteins (Rb) and the E2F transcription factors (E2F) have been reported in Arabidopsis, only a small number of core cell cycle genes were functionally examined in monocots. Rice, as a model plant species of monocots, is suitable for the identification of core cell cycle genes because of the availability of its whole genome sequences. In this study, we searched for the predicted core cell cycle genes from rice genome, and examined their expression patterns in various rice tissues and the regulation of their expression by cytokinin and auxin, and then determined the role of some candidate genes during rice development.In order to obtain the putative core cell cycle genes in rice, blast searches were conducted against the rice database using the Arabidopsis core cell cycle genes published as query sequences. Ninety putative core cell cycle proteins were identified and they belong to cyclin, CDK, E2F, CKI, CKS, Rb and Wee families, respectively. Phylogenetic and structural analysis indicated that both the number and the structure of the core cell cycle regulators are relatively conserved between the rice and Arabidopsis genomes.To understand the fuctions of rice core cell cycle genes, their expression patterns in rice were investigated by semi-quantitative RT-PCR. The tissues include roots, young leaves, spikelets, seeds (0, 1, 3, 6 days after pollination or DAP), endosperms (9, 12 and 15 DAP), and embryos (9 and 15 DAP). The results indicated that the expression of most of the core cell cycle genes are spatially regulated, and they can be divided into five classes based on their expression patterns. The expression patterns of some closely related core cell cycle genes are similar, suggesting functional redundancy of these core cell cycle genes.In situ hybridization were conducted to study the transcript localization of the core cell cycle genes in rice. Four genes including Orysa;CycA2;1, Orysa;CycB2;2, Orysa;CycU4;4 and Orysa;CDKG;1 were randomly selected and the results suggested that they are temporally and spatially regulated. Also, this data supports the expression profiles underlied by the RT-PCR analysis. Following auxin or cytokinin treatment, the expression of some core cell cycle genes was either upregulated or downregulated, indicating that auxin and/or cytokinin regulate the expression of these core cell cycle genes.Seven core cell cycle genes such as Orysa;CycA2;1, Orysa;CycB1;1, Orysa;CycB2;2, Orysa;CDKE, Orysa;DEL1, Orysa;DP1 and Orysa;E2F4 were chosen to study their functions in rice development by genetic transformation. The results showed that Pubi::CycB1;1RNAi transgenic rice plants displayed aberrant phenotypes, such as lower seed-setting rate and abnormal seed morphology. Histological analysis indicated that abortive endosperm while larger embryo with disturbed morphology and structures to some extent setted in the abnormal seeds compared with the control one. Further analysis showed that endosperm-specific knockdown of Orysa;CycB1;1 produced similar abnormal seeds, indicating that Orysa;CycB1;1 regulated the development of endosperm directly. Statistical analysis showed that the enlarged embryo size in Pubi::CycB1;1RNAi plants was due to the increased cell size.The expression patterns of Orysa;CycB1;1 was confirmed using semi-quantitative RT-PCR, qRT-PCR, in situ hybridization and histological staining of PCycB1;1::GUS transgenic rice seeds. The results indicated that Orysa;CycB1;1 was highly expressed in tissues or cells which divide rapidly, and its transcripts were identified in endosperm and embryo.Expression analysis of the seed development-related genes in PGluA-1::CycB1;1RNAi transgenic rice endosperm indicated that the transcript levels of FIE and GW2 were stronger than that of control. Microarray analysis revealed that many genes involved developmental processes, morphogenesis and cell wall organization were significantly upregulated or downregulated in transgenic embryos. It was suggested that these gene expression might be regulated by Orysa;CycB1;1 during seed deveopment.In conclusion, we have conducted genome-wide identification and expression analysis of rice core cell cycle genes, and provided the evidences that Orysa;CycB1;1 plays the important roles in endosperm and embryo development in rice. Our current results might provide basic information to understand the mechanism of cell cycle regulation and the functions of the core cell cycle genes in rice.