Overexpression of Atriplex canescens Flavanone 3-Hydroxylase (AcF3H) Enhances Salt and Drought Tolerance in Arabidopsis thaliana via Flavonoid-Mediated ROS Homeostasis

Flavonoids play critical roles in plant adaptation to abiotic stress by acting as potent antioxidants that regulate reactive oxygen species (ROS) homeostasis. In Atriplex canescens (Pursh) Nutt., a halophytic shrub well-adapted to saline and arid environments, transcriptomic analyses revealed that salt stress induces strong upregulation of flavanone 3-hydroxylase (F3H), a key enzyme in the flavonoid biosynthetic pathway. However, the functional role of AcF3H in stress adaptation remains poorly understood. Here, we cloned the AcF3H gene from A. canescens and generated transgenic Arabidopsis thaliana (L.) Heynh. lines constitutively overexpressing this gene. Overexpression of AcF3H significantly enhanced flavonoid accumulation, as confirmed by DPBA staining and total flavonoid quantification, and selectively upregulated the expression of downstream biosynthetic genes AcDFR and AcANS, which encode the dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), respectively. Under salt and drought stress, transgenic lines exhibited improved root elongation, increased shoot and root biomass, and higher relative water content compared to wild-type plants. Mechanistic investigations revealed that AcF3H overexpression led to reduced H2O2 accumulation and lower plasma membrane permeability under stress conditions, indicating enhanced antioxidative capacity and cellular membrane stability. These results suggest that AcF3H confers enhanced tolerance to abiotic stresses by promoting flavonoid-mediated ROS homeostasis. Our findings highlight AcF3H as a promising genetic target for engineering salt- and drought-tolerant crops.