Generating Transgenic Arabidopsis Expressing Redox-Sensitive Green Fluorescent Protein

• Sarah Ding, Plant Science, University of Delaware

• Jung-youn Lee, Plant and Soil Sciences, University of Delaware

ROS species such as hydrogen peroxide (H2O2) are known to function as signaling molecules crucial to cellular metabolism, programmed cell death, and environmental response in plants. Under stress, ROS accumulates to phytotoxic levels, causing oxidative stress and altering the redox state in the cell. Factors affecting redox state within plant cells include the glutathione buffer, antioxidants, ROS, and more, each controlled by a variety of genes. Using the redox-sensitive modified tag protein roGFP2 fused to endogenous plasmodesma-localized protein PDLP5, the redox state of plasmodesmata may be measured in response to ROS. Wild-type Arabidopsis thaliana were transformed by ‘floral dipping,’ resulting in successful infiltration of the gene construct into the developing seed germplasm. Inflorescences were dipped twice in a solution containing transformed Agrobacterium containing the gene construct PDLP5-roGFP2 in the span of 2 weeks. Seeds were screened on 5% BASTA plates, and localized roGFP2 fluorescence at plasmodesmata was expressed. Transgenic organisms were successfully generated, and expression of GFP was confirmed using fluorescent confocal microscopy, proving that roGFP2 can be effectively expressed within plasmodesma channels. Transgenic plants carrying PDLP5-roGFP2 are used to elucidate the functions and pathways of various genes involved in plant immunity and stress response under redox state changes caused by generation of ROS species. The precise functions of these targeted genes may be monitored by measuring changes in redox state using roGFP2 in mutants.