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

Abstract

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 (H 2 O 2 ), 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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-H 2 O 2 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–H 2 O 2 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.

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