5-Aminolevulinic acid improves strawberry salt tolerance through a NO–H2O2 signaling circuit regulated by FaWRKY70 and FaWRKY40

Introduction

5-Aminolevulinic acid (ALA) is an essential biosynthetic precursor of tetrapyrrole compounds, naturally occurring in all living organisms. It has also been suggested as a new plant growth regulator. Treatment with ALA promotes strawberry Na⁺ homeostasis under salt stress. Regulation of this process requires the signaling molecules nitric oxide (NO) and hydrogen peroxide (H₂O₂), but the specific signaling cascade and transcriptional regulatory mechanism have not previously been characterized.

Objectives

Our work focused on the dissection of the NO and H₂O₂ signaling cascade and transcriptional regulatory mechanism by which FaWRKY70-FaWRKY40 participated in ALA-improved Na⁺ homeostasis and salt tolerance of strawberry.

Methods

It was preliminarily confirmed by transcriptome and RT-qPCR that FaWRKY40 and FaWRKY70 participated in ALA-induced salt tolerance of strawberry. Two WRKY transcription factors overexpressed in woodland strawberry as well as tobacco were used to identify the gene functions in salt tolerance. Yeast one-hybrid (Y1H), β-glucuronidase (GUS), dual luciferase reporter (DLR) and electrophoretic mobility shift assays (EMSA) were used to verify the interaction with the target gene.

Results

ALA induced NO and H₂O₂ production, which formed a signaling circuit reciprocally regulated by FaNR1 and FaRbohD expression to coordinate Na⁺ homeostasis. FaWRKY40 was shown to act as a positive transcription factor in this pathway: FaWRKY40 overexpression improved salt tolerance in woodland strawberry and tobacco, whereas FaWRKY40 RNA interference increased plant salt injury. FaWRKY40 bound to the promoters of FaRbohD, FaNHX1, and FaSOS1 to promote root H₂O₂ generation and Na⁺ reallocation. Conversely, FaWRKY70, a negative WRKY transcription factor, was found to increase salt sensitivity by inhibiting expression of FvWRKY40, FvNR1, and FvHKT1. ALA inhibited FaWRKY70 but increased FaWRKY40 expression, coordinating the regulation of NO-H₂O₂ signaling and Na⁺ homeostasis when strawberry was stress by salinity.

Conclusion

ALA inhibits NaCl-stimulated FaWRYK70 expression, relieving the transcriptional inhibition of its downstream targets. The NO–H₂O₂ signaling circuit can then initiate mechanisms such as Na⁺ exclusion, vacuolar sequestration, and removal of Na⁺ from the xylem sap, limiting Na⁺ accumulation in the leaves and promoting Na⁺ homeostasis and plant salt tolerance.