Flavonol Regulation of Root Development in Arabidopsis

The signaling pathways controlling lateral root and root hair initiation and elongation are only partially understood. We are testing the role of reactive oxygen species (ROS) and endogenous antioxidants in modulating these developmental programs. We used confocal microscopy to visualize the accumulation of ROS using a generic ROS sensor, H₂DCFDA, and an H₂O₂-specific sensor, PeroxyOrange1. ROS is at high levels in root epidermal tissues from which root hairs form and in the epidermal and cortical layers overlaying emerging lateral roots. We compared the ROS pattern to that of flavonols, one family of potent ROS scavengers, using the fluorescent probe, DPBA. Kaempferol accumulates in dividing cells of lateral root primordia, quercetin is in tissue from which primordia form, while both flavonols are at low levels in tissues overlaying primordia. Flavonols are also largely excluded from the epidermal tissues from which root hairs initiate. The fluorescence of a CHS-GFP reporter to visualize location of the first enzyme in flavonol synthesis overlays the DPBA signal. The localization of flavonols is inversely correlated with ROS concentrations, consistent with flavonols acting as antioxidants. To test the function of flavonols and ROS in root hair formation, we used Arabidopsis mutants defective in flavonol biosynthetic enzymes and found that tt4 and tt7 mutants, which produce no flavonols or only kaempferol, respectively, have more root hairs than Col-0. In contrast, the omt1 mutant, which does not produce isorhamnetin, has similar root hair numbers to Col-0, consistent with quercetin being the active flavonol in regulating root hair formation. ROS probe signals are elevated in the tt4 mutant, consistent with the antioxidant function of flavonols. Chemical complementation with a flavonol precursor decreased root hair number and ROS accumulation in tt4. These results suggest flavonols regulate root hair development by modulating ROS levels. Support comes from NSF IOS Grant 1558046.