Scientific Abstract
Summary of the Proposed Project.
Reactive oxygen intermediates (ROI) control many different processes in plants. However, they are also toxic molecules capable of injuring plant cells. How this conflict is resolved in cells is largely unknown. Nonetheless, it is clear that the steady-state level of ROI in cells needs to be tightly regulated. Our goal is to identify and characterize regulatory transcripts directly involved in controlling ROI metabolism in plants. We propose a unique approach of using knockout lines, deficient in ROI-scavenging enzymes, to induce the accumulation of ROI in specific cellular compartments, in the absence of external stimuli or stress. We hypothesize that knockout plants with suppressed capability to scavenge ROI will accumulate ROI and activate their ROI signal transduction pathway. The study of gene expression in these plants will therefore enable us to identify and characterize some of the key regulatory genes that control ROI accumulation in plants. Moreover, our approach will enable us to simplify the complex interactions that exist between ROI and many different biotic and/or abiotic signaling pathways, and to isolate and characterize signal transduction transcripts directly involved in regulating ROI metabolism in plants. Based on our preliminary results we hypothesize that zinc-finger proteins play a central role in the ROI signal transduction pathway of plants. We will determine how they control ROI metabolism and how they fit into the hierarchy of ROI-response genes. Knockout Arabidopsis lines with suppressed capability to scavenge ROI will be used to induce an internal oxidative stress in plants under controlled conditions (using a time-course design). Microarray analysis coupled with physiological and biochemical measurements will be used to identify regulatory transcripts induced/suppressed in cells upon ROI accumulation. Seedlings of transgenic plants constitutively expressing zinc-finger proteins and other transcription factors, induced/suppressed in cells upon ROI accumulation, will be screened for tolerance/sensitivity to oxidative stress on agar plates. The function of specific transcripts, identified by our screen as capable of altering the ROI-tolerance of plants, will be determined in transgenic plants and knockout lines. A genetic screen to identify ROI-sensing components will be developed.
Broader Impacts of the Proposed Project.
Data, methods, and biological material will be made available to the public as soon as they are confirmed. Up to 20 undergraduate students, including minorities, will be recruited into the project and introduced to modern research techniques in plant biology, including microarray analysis and mutant characterization. An online course for undergraduate students will be developed to share knowledge and enhance awareness to ROI and the important role they play in plants and other organisms. A high school teaching kit that includes a video presentation and several basic experiments demonstrating the importance of ROI to plant protection will be developed. Public access to the project will be ensured through a website specifically developed for the project. ROI are implicated in a diverse array of biological processes. The ROI-sensitive/tolerant lines, the database of ROI-associated transcripts and the flow-chart of ROI response in plants (developed during the course of this study), could be used by many different research groups and scientists to determine to what extent and in what manner ROI are involved in the biological process/pathway they are studying. They would therefore serve as a valuable resource and enhance our overall understanding of ROI function in plants during normal metabolism, as well as during periods of environmental stress.