Abiotic Stress Function Analysis Of Maize Transcription Factor ABP9 In ABA Signaling

Low temperature, drought, and high salinity are common stress conditions that adversely affect plant growth and crop productivity. It is well known that in China there are lots of droughty and semiarid land, accounting for 38% of the arable land, and great acres of saline soil need increasing productivity and reclamation. In addition, low/freezing temperatures restrict the planting and harvest of crops in a year-round manner. Therefore, enhancing the plant ability against environmental stresses is critical for increasing the yield of crops.Oxidative stress is the common mechanism by which abiotic stresses affect plant growth and development. Under adverse environmental conditions, such as drought, high salinity and low temperature, the endogenous level of plant hormone ABA (Abscisic Acid) rapidly increases which leads to excess production of reactive oxygen species (ROS), resulting in the formation of oxidative stress that causes irreversible injuries to plants. To scavenge excess ROS and reduce its toxic effects, plants have evolved an antioxidant system which is up-regulated under stress conditions and correlated well with enhanced tolerance. However, due to the poor capability for induction of the various enzymes of the antioxidant defense system, the degree of the enhancement is limited. Therefore, a more enhanced anti-oxidant ability is required for improving plant stress tolerance.As a part of the anti-oxidant system of Maize, Cat1 gene plays an indispensable role in scavenging excess cellular H2O2. Our previous studies have demonstrated that the expression of Cat1 is ABA-inducible and is mediated by ABRE2 cis-element in its promoter. The transcription factor ABP9 that specifically interacts with ABRE2 has been cloned. Given that ABRE motif is located in the promoters of many stress-induced genes, it will be interesting to know if ABP9 can trigger the expression of a battery of ABA-related genes and contribute to the reduction of ROS level. My research thesis focuses on addressing these questions by analyzing the role of ABP9 in ABA and ROS signaling transduction pathway in transgenic Arabidopsis plants. The research content and the main conclusions are as follows:1. ABP9 constructs were made, pBI121-35Sp-ABP9-NOSter and pCHF3-35Sp- ABP9Dominant Negative-NOSter for stable transformation of Arabidopsis, respectively.2. ABP9 and ABP9DN transgenic Arabidopsis plants were obtained through agro-bacterium–mediated transformation via vacuum infiltration. Identification of transgenic plants by PCR was done on 62 and 41 kanamycin resistant T1 plants and revealed that 75.8% and 82.9% of them were PCR positive.3. Five homozygous ABP9 transgenic lines were used to analyze the expression of ABP9 and other stress-related genes by RT-PCR. The results showed that ABP9 gene was over-expressed and its expression level in the five lines was different, with 5# line the strongest. The expression of ABA synthesis-related genes nced3 and aba1 were significantly down-regulated related. While abi1 gene was up-regulated comparing to their expression in the wild-type control.4. ELISA method was employed to quantify the level of abscisic acid in plants. The results showed that the expression of ABP9 gene in Arabidopsis led to about 30%~40% decrease of abscisic acid level in transgenic plants, compared with that in non-transgenic ones. ABA tolerance assays of ABP9 transgenic Arabidopsis plants showed that ABP9 overexpression affected endogenous ABA level, and the transgenic plants are sensitive to ABA in terms of their growth and development..5. ESR method was employed to quantify the level of reactive oxygen species in plants. The results showed that the expression of ABP9 gene in Arabidopsis led to about 78.9%~57.7% decrease of ROS amount in transgenic plants, compared with that in non-transgenic ones. By using 2',7'-dichlorofluorescin diacetate(H 2DCF-DA) we determined the reactive oxygen species(ROS) production in guard cells. The results, combined with ESR spectrum, strongly suggest that ABP9 regulated many downstream ROS related genes'expression and increased plants'ability to scavenge ROS.6. The result suggests that ABP9 gene plays an important role in plant growth and development.In conclusion, transcription factor ABP9 can specifically bind to ABRE and plays an important role in ABA and ROS signaling. This Indicates that ABP9 is a good candidate for improving crop performance under environmental stresses.