Overexpression of soybean flavonoid 3′-hydroxylase enhances plant salt tolerance by promoting ascorbic acid biosynthesis

Schematic model of F3′H-mediated promotion of 3′-hydroxylated flavonoid compounds and AsA accumulation, thereby conferring plant salt tolerance. Firstly, F3′H enhances the salt tolerance of soybean seeds during germination by catalyzing the synthesis of the 3′-hydroxylated flavonoid compounds in the seed coat. Secondly, F3′H and VTC1 competed for binding to the CSN5B protein to inhibit the degradation of VTC1 by CSN5B, which promoted the biosynthesis of AsA to enhance the ability to scavenge reactive oxygen species and the salt tolerance of seedlings. • We identified the flavonoid 3′ hydroxylase gene ( GmF3′H ) in soybean, which confers salt tolerance during seed germination and seedling in soybean and Arabidopsis thaliana . • This study demonstrated that functionally conserved F3′H can competitively bind to photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) with GDP-Man pyrophosphorylase (VTC1) which is the ascorbic acid (AsA) biosynthetic enzyme for seedling salt stress tolerance in plants. • Discovery and characterization of F3′H -based salt tolerance pathway will provide new insights for understanding of plant salt tolerance mechanism and offer an important avenue/approach for improving broad-spectrum salt tolerance in crop breeding. Salt stress is a major cause of crop loss. Soybean ( Glycine max), a globally vital legume crop, faces mounting yield constraints due to soil salinization. It is known that the flavonoid biosynthesis pathway involving flavonoid 3′-hydroxylase (F3′H) plays an important role in salt tolerance. However, the precise molecular basis of F3′H-mediated salt tolerance remains inadequately characterized. This study aimed to elucidate the function and explore the pleiotropic molecular basis of F3′H protein in soybean salt tolerance. Innovation on elite new crop varieties facilitates breeding and production applications on salt tolerance. We employed CRISPR/Cas9-mediated knockout and Agrobacterium -based overexpression to generate GmF3′H allelic variants and ectopic expression in soybeans. Sanger sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to confirm the specificity of gene editing and quantify expression levels in overexpression transgenic plants, respectively. As well as Subcellular localization analysis, Yeast two-hybrid (Y2H) assay, LUC activity assay and plant physiological measurements were carried out to elucidate the F3′H-mediated salt tolerance molecular basis in plants. In this study, we identified the flavonoid 3′ hydroxylase gene ( GmF3′H ) in soybeans, which as a master regulator of salt stress adaptation during seed germination and seedling stages in both soybean and Arabidopsis thaliana . Furthermore, our study revealed that the evolutionarily conserved F3′H protein competitively binds to the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) and disrupts its interaction with GDP-mannose pyrophosphorylase 1 (VTC1), a key enzyme in ascorbate biosynthesis. This competitive inhibition redirects metabolic flux toward the L-galactose pathway, leading to an increase in ascorbic acid (AsA) biosynthesis. The enhanced AsA production subsequently improves seedling salt stress tolerance in plants by maintaining redox homeostasis through ROS scavenging. The discovery and characterization of F3′H-mediated salt tolerance provide a crucial framework for the genetic improvement of crops. This work provides new insights into plant salt stress tolerance and develops innovative strategies to enhance broad-spectrum salt tolerance, a crucial aspect for ensuring food security in crops.