The clock component LHY1b negatively regulates alkaline stress by repressing AOX1-mediated oxidative responses in soybean

Increasing the ability of crop plants to maintain productivity in saline-alkaline lands is an absolute requirement for feeding the growing population. However, a shortage of knowledge about plant alkaline tolerance restricts breeding of salt-tolerant crops. Here, we demonstrate that a homolog of the circadian clock component LATE ELONGATED HYPOCOTYL (LHY) negatively regulates reactive oxygen species (ROS) homeostasis and time-gates oxidative stress responses under NaHCO3 conditions in soybean (Glycine max). Yeast one-hybrid assays followed by chromatin immunoprecipitation coupled with quantitative PCR, electrophoretic mobility shift assays, and dual-luciferase reporter assays revealed that LHY1b binds directly to the promoter of Alternative Oxidase 1 (AOX1). Through immunoblotting using AOX1 antibody, we demonstrated that AOX1 accumulates considerably in lhy1b under NaHCO3 conditions. LHY1b strongly inhibits AOX1 transcription under normal growth conditions. However, this inhibition is removed under alkaline stress, allowing the expression of AOX1 and restricting the generation of ROS. Knockdown of AOX1 in lhy1b renders this mutant more sensitive to alkaline stress. Moreover, we determined that the naturally occurring LHY1bH2 allele is associated with alkaline tolerance by phenotyping 559 sequenced soybean accessions. Our study demonstrates a molecular link between clock components and alkaline tolerance and provides a valuable target gene for breeding alkaline-tolerant soybean varieties.