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

Introduction

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.

Objectives

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.

Methods

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.

Results

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.

Conclusion

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.