Isolation And Characterization Of Rice Genes Involved In Embryo Development

Angiosperms are considered as a monophyletic group comprising two classes, the monocotyledons and the dicotyledons. Higher plants develop from the single-cell zygote, and distinct embryogenesis differentiations exist between dicotyledons and monocotyledons. More information on embryogenesis have been obtained in dicotyledons than monocotyledons. In this study, we analysed the expression of genes may involve in rice embryogenesis, Semi-quantitative reverse transcriptase PCR (Semi-RT-PCR) was performed on cDNAs prepared from different tissues. Then, we focused on the expression and functional analysis of OsWOX2 and OsWOX4. Both genes coexpressed in most of the lateral organ primordia, the young leaves, and the developing seeds without adaxial/abaxial polarity, neither of the isolated rice WOX orthologues displays an organizing center-type expression pattern in the vegetative SAM as in Arabidopsis. We tried to provide evidences for understanding their effects in rice embryogenesis through down-regulation and overexpression plants. The main results in this study are as follows:1. We detected the expression of twelve kinds of genes involved in rice embryogenesis, found that there are higher similarity gene expression patterns between Arabidopsis and rice, suggesting that there are similar functions among these genes in embryogenesis between two species. Auxin plays an important role in Arabidopsis pattern formation. The expression of most of the genes involved in rice embryogenesis were induced by IAA, indicating that auxin might be necessary for rice embryogenesis. 2. We have identified twelve members of the WUS-related homeobox (WOX) family genes from rice genome. Most of the OsWOX contain a WUS-box toward the amino terminus in addition to the homeodomain toward the carboxyl-terminus end. The phylogeny based on the HD amino acid sequence of the WOXs was established in the plant species including Arabidopsis, Oryza sativa, Zea mays, and Brachypodium distachyon. Phylogenic analysis of WOX proteins showed that OsWOXs were highly conserved with ZmWOXs and BdWOXs. And, most of the OsWOX expressed in active cell division regions, such as shoot apical meristem, root apical meristem, inflorescence through Semi-quantitative RT-PCR or RNA in situ hybridization.3. Phylogenic analysis of OsWOX proteins showed that rice WOX2 was highly conserved with ZmWOX2A and ZmWOX2B, with homeodomain sharing 73.85% similarity to Arabidopsis WOX2 homeodomain. To study the subcellular localization of OsWOX2, the full-length of OsWOX2 coding sequence was translationally fused to the GFP-coding sequence under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and the constructs were introduced into the onion cells using the particle bombardment method. We found that the OsWOX2-GFP fusion proteins were located throughout the cells by examination with confocal microscopy.In situ hybridization experiments with a gene-specific region of the rice WOX2 cDNA as the probe revealed that the expression pattern of OsWOX2 is localized in the shoot apical meristem, root apical meristem, and young leaf primordia. Plants with the down-regulation genes produced aberrant twisted leaves displaying knotted outgrowth, larger mature blades, and disorganized veins. Moreover, the tiller buds on the elongated upper internodes were induced when the primary stem begin to differentiate their own panicles. The development of the pollen and embryo sac were destructed.4. OsWOX4 was highly conserved with BdWOX2 and ZmWOX2, with homeodomain sharing 80.00% similarity to Arabidopsis WOX4 homeodomain. OsWOX4 transcripts were detected in young leaf primordia and the developing seed. Higher levels of OsWOX4 transcripts are apparent at the shoot apex and the tip of the root in the mature embryo. Transgenic plants with down-regulation were dwarf. Tansgenic brades were narrow than the wild ones. Also, number of the spikelets was decreased, and the endosperm size was reduced in transgenic plants.