Down-regulation of free riboflavin content induces hydrogen peroxide and a pathogen defense in Arabidopsis

Riboflavin mediates many bioprocesses associated with the generation of hydrogen peroxide (H2O2), a cellular signal that regulates defense responses in plants. Although plants can synthesize riboflavin, the levels vary widely in different organs and during different stages of development, indicating that changes in riboflavin levels may have physiological effects. Here, we show that changing riboflavin content affects H2O2 accumulation and a pathogen defense in Arabidopsis thaliana. Leaf content of free riboflavin was modulated by ectopic expression of the turtle gene encoding riboflavin-binding protein (RfBP). The RfBP-expressing Arabidopsis thaliana (REAT) plants produced the RfBP protein that possessed riboflavin-binding activity. Compared with the wild-type plant, several tested REAT lines had > 70% less flavins of free form. This change accompanied an elevation in the level of H2O2 and an enhancement of plant resistance to a bacterial pathogen. All the observed REAT characters were eliminated due to RfBP silencing (RfBPi) under REAT background. When an H2O2 scavenger was applied, H2O2 level declined in all the plants, and REAT no longer exhibited the phenotype of resistance enhancement. However, treatment with an NADPH oxidase inhibitor diminished H2O2 content and pathogen defense in wild-type and RfBPi but not in REAT. Our results suggest that the intrinsic down-regulation of free flavins is responsible for NADPH oxidase-independent H2O2 accumulation and the pathogen defense.