The SmRAP2-4-SmAMR1-SmEF1A module regulates plant salt tolerance in Salix matsudana

Abstract

F-box proteins are important components of the SCF-E3 ubiquitin ligase complex, specifically regulating target protein abundance and participating in plant growth, development, and stress responses. In this study, an F-box gene responsive to salt stress, SmAMR1 , was cloned from Salix matsudana Koidz. It was 1209 bp long and encoded 402 amino acids. The SmAMR1 -overexpressing tobacco exhibited the worst state under salt stress. After 12 d of salt treatment, the survival rates of the transgenic lines were 40.90% and 49.99% lower than the WT lines, respectively. While the MDA content and REL values of the SmAMR1-transgenic tobacco plants were, on average, 1.74- and 1.23-fold higher than those of the WT, indicating that SmAMR1 can significantly reduce plants’ salt tolerance. The Y2H and LCA assays revealed that SmAMR1 can bind to SmSKP1-SmCUL1 and form the SCF SmAMR1 complex, which further binds to the target protein of SmEF1A. A Western blot assay revealed that the complex can mediate the ubiquitination and degradation of SmEF1A. Moreover, SmEF1A was cloned and introduced into tobacco plants, and the transgenic lines demonstrated a better growth state under salt stress, with improved morphological and physiological indexes compared to the WT plants. In addition, the Y1H and dual-LUC assays revealed that an AP2/ERF transcription factor, SmRAP2–4, can bind to the GCC box-like element on the SmAMR1 promoter and negatively regulate SmAMR1 expression. In summary, the SmRAP2–4 - SmAMR1 - SmEF1A module acted as a novel pathway and significantly participated in the salt tolerance of S. matsudana plants.