Flavonol antioxidants reduce high temperature-induced Reactive Oxygen Species (ROS) and enhance tomato pollen performance

In angiosperms, the pollen tube delivers sperm cells from the pollen grain to the female gametophyte leading to fertilization and seed development. Reactive Oxygen Species (ROS) are signaling molecules that are necessary for pollen tube germination and elongation however, we have observed that temperatures above the optimal increase ROS accumulation to levels that impair pollen performance. To observe the temperature induced increases in ROS with high temporal and spatial resolution, we transformed Solanum lycopersicum (tomato) with Hyper7, a H2O2 biosensor. Utilizing this line, we are monitoring spatial and temporal ROS dynamic changes during pollen tube growth and tip rupture under control and elevated temperatures in tomato pollen. The anthocyanin-reduced (are) mutant, has a defect in the flavanone 3 hydroxylase (F3H) gene, leading to reduced flavonols. We found that under high temperature VF36 (Wild type) and are both had impaired pollen viability, germination, pollen tube growth, and increased ROS levels, with greater effects in are. The genetic complementation of are and overexpression of the F3H gene reduced temperature induced ROS accumulation in pollen grains and tubes and enhanced pollen performance, with F3H overexpression fully protecting pollen from high temperature stress. The thermotolerant tomato cultivar Tamaulipas exhibited enhanced ROS homeostasis in pollen tube and improved seed set under elevated temperatures. Tamaulipas has increased abundance of transcripts encoding flavonol biosynthetic enzymes in pollen tube. We observed elevated flavonol levels in pollen tubes and pistils suggesting that natural variation that increases flavonol levels increases reproductive success in tomato. We are also testing the hypothesis that elevated temperatures during pollen development may transiently increase flavonol levels to protect pollen from heat stress. These results provide insights into mechanisms by which high temperature impairs pollen germination and tube growth and how flavonols prevent these effects, enhancing our understanding of how flavonols and ROS can influence thermotolerance.