Mechanism Analysis Of Resistance To Oxidative Stress In Bcl-2 Transgenic Rice And Development Of Root Border Cells

Environmental stresses are the main factors limiting plant growth and development, which seriously constraint the yields and qualities of crops. It has been an important strategy to engineer agricultural crops to improve stress resistance. In this study, the molecular mechanism of resistance to oxidative stress in rice was investigated by focusing on programmed cell death (PCD) and root border cell ( RBC ) development.1. Functional analysis of Bcl-2 in transgenic rice in response to H₂O₂-induced PCDProgrammed cell death (PCD) is a genetically controlled cell death processes during plant development or in response to environmental stresses. Previous studies have revealed that ectopic expressions of some animal anti-apoptosis genes can inhibit PCD and improve resistance to biotic and abiotic stresses in plant. However, very little is known about the underlying molecular mechanisms.Here, we transferred the human anti-apoptosis gene Bcl-2 into rice by Agrobacterium tumefaciens-mediated transformation, and obtained five lines of 35S::Bcl-2 homozygous transgenic rice. Northern blot confirmed the expression of Bcl-2 in transgenic lines. In contrast to wild type, H₂O₂-inhibition of seed germination, chlorophyll retention, root elongation and root tip viability was significantly alleviated in the transgenic rice, suggesting that over-expression of Bcl-2 could enhance the resistance to H₂O₂ toxicity.The results of DNA laddering and TUNEL test indicated that H₂O₂ induced cell death accompanied by typical hallmarks of PCD, while Bcl-2 could promote H₂O₂ tolerance in transgenic plants via inhibition of PCD.To investigate PCD associated genes in plant, we found 4 OsVPEs and 7 OsMCs in rice genome by sequence alignment and function domain analysis. The expressions of these two kinds of caspase-like genes were analyzed by semi-quantitative RT-PCR. The expression analysis showed that the expression of OsVPE-1 in root tips was significantly increased after 6, 8, 12 and 24h of H₂O₂ treatment. The level of OsVPE-2 transcript accumulated after 2h and lasted till 12h treatment, and decreased to the level of controls after 24h. The results suggested that H₂O₂-induced PCD might be regulated through vacuolar pathway. However, in Bcl-2 transgenic plants, the expression of VPE did not change after H₂O₂ treatment, implying that Bcl-2 might repress H₂O₂-induced PCD in rice by inhibiting expression of VPE.2. Development regulations of root border cells in rice.More and more evidences have revealed that root border cells, whose development is genetically regulated, are biologically viable (>90%) in the majority of higher plant species, and take multi-defense roles during root growth and development. Hence, studies on developmental regulation and biological functions of root border cells have attracted the attention of many researchers. However, the root border cells production, number and viability in rice are little known.During seed germination, the formation of the first root border cell was observed almost synchronously with root tip emergence. Our results indicated that each rice root tip owns about 1500 root border cells, and 95% of which was viable by cell counting and viability assay. Further observation showed that root tips could reproduce another set of root border cells in 36 hours after removal of existing border cells. Root tips could produce much more root border cells when stimulated by some phytohormones such as BR, GA₃, IAA and KT. It suggested that production and development of the root border cells was regulated by multiple phytohormones. The activity of PME in root caps was estimated on different developmental stages and various time points after removing root border cells in rice root. The results showed closely correlation between root border cell development and PME activity in root caps. The gene OsPME-1(Os04g0458900) was cloned from rice, which encodes 568 amino acids and contains two conserved functional domains (PMEI and PME domains). The result of semi-quantitative analysis showed that OsPME-1 expression pattern was consistent with the production of root border cells.Four plant expression vectors (pCAMBIA13011-Ospme, pCAMBIA13011-anti-Ospme, pCAMBIA13011- Ospmei-Domain and pCAMBIA13011-Ospme-Domain) were constructed and transformed into rice by Agrobacterium tumefaciens-mediated transformation. The transgenic rice plants were verified by using hygromycin selection and RT-PCR test. Until now, the T2 generation of transgenic rice has been obtained, and detailed study of the function of OsPME-1 in transgenic plants is now in progress.