Quantitative ROS bio-reporters: a robust toolkit for studying biological roles of reactive oxygen species in response to abiotic and biotic stresses.

While the accumulation of reactive oxygen species (ROS) through spontaneous generation or as the by-products of aerobic metabolism can be toxic to plants, recent findings demonstrate that ROS act as signaling molecules that play a critical role in adapting to various stress conditions. Tight regulation of ROS homeostasis is required to adapt to stress and survive, yet in vivo spatiotemporal information of ROS dynamics are still largely undefined. In order to understand the dynamics of ROS changes and their biological function in adapting to stresses, two quantitative ROS transcription-based bioreporters were developed. These reporters use ROS-responsive promoters from RBOHD or ZAT12 to drive green fluorescent protein (GFP) expression. The resulting GFP expression is compared to a constitutively expressed mCherry that is contained on the same cassette with the ROS-responsive promoter: This allows for the generation of ratiometric images comparing ROS changes (GFP) to the constitutively expressed mCherry. Both reporters were used to assess ROS levels to oxidative stress, salt stress, and the pathogen defense elicitor flg22. These bioreporters showed increases in the ratio values of GFP to mCherry signals between 10 and 30min poststress application. Such stress-associated ROS signals correlated with the induction of abiotic/biotic stress responsive markers such as RbohD, ZAT12, SOS2 and PR5 suggesting these ROS bioreporters provide a robust indicator of increased ROS related to stress responses. Based upon the spatiotemporal response patterns of signal increase, ZAT12 promoter-dependent ROS (Zat12p-ROS) bioreporter appears to be suitable for cellular mapping of ROS changes in response to abiotic and biotic stresses.