Optimizing a Luciferase-Based Tool for Studying the Effects of Fatty Acid Desaturase 7 on Singlet Oxygen Accumulation in Arabidopsis thaliana

In plants, reactive oxygen species (ROS) are generated as a byproduct of normal metabolism, as well as in response to adverse conditions such as light stress, extreme temperatures, and exposure to pests and pathogens. Singlet oxygen (1O2) is a ROS that is formed during photosynthesis in photosystem II (PSII) of the chloroplasts. Levels of 1O2 and other ROS are tightly controlled in healthy plants, but some studies suggest that levels of fatty acid desaturase (FAD) activity in Arabidopsis thaliana can influence constitutive and/or stress-responsive ROS accumulation. In this study, a luciferase-based reporter gene that is selectively stimulated by 1O2 (AAA-ATPase: Luc) was used to compare 1O2 levels in wild-type Arabidopsis plants (Col-0) and a mutant line with impaired fatty acid desaturation (fad7-1). The goals of this study were to establish a suitable method for measuring luminescence generated by the 1O2 –responsive reporter gene, and to use this method to see if alterations in fatty acid desaturation have an impact on ROS accumulation in the chloroplast. The AAA-ATPase: Luc reporter gene was successfully introduced into the two genetic backgrounds, Col-0 and fad7-1, and reporter gene activity was measured using a luminometer-based assay of leaf extracts, a plate reader-based assay of intact leaf discs or extracts, and a camera-based system for imaging intact rosettes (a Lumina XR). Our results suggested that the luminometer-based assay had greater sensitivity than the other two approaches. We also found that, in both genetic backgrounds, activity of the reporter gene increased in response to high light and rose bengal (a chemical elicitor of 1O2), but not in response to aphid infestation. Although fad7-Luc and Col-Luc were similar in their response to these three treatments, our data suggested that constitutive levels of reporter gene activity were higher in the fad7-1 background than in Col-Luc. Potentially, decreased fatty acid desaturation in fad7-1 may promote higher constitutive levels of 1O2