uORF-regulated transcription factor MsSPL12 mediates alfalfa drought tolerance via activating ABA biosynthesis

Abstract

Drought stress severely restricts the growth and yield of alfalfa (Medicago sativa L.), and ABA plays a vital role in plant drought tolerance. Here, we report that a transcription factor MsSPL12, induced by both drought and ABA treatment, mediates drought tolerance in alfalfa. Overexpression of MsSPL12 enhanced drought resistance and ABA sensitivity in alfalfa plants, while suppression of its expression led to the opposite phenotypes. Population genetic analysis across Medicago species revealed a SNP in the 5’UTR of MsSPL12 that converts the strong repressive Type I upstream open reading frame (uORF) into a weaker Type II. Disruption of the Type I uORF12 by using the CRISPR/Cas9 system markedly elevated the translation efficiency of MsSPL12, leading to enhanced ABA sensitivity and improved drought resistance in two alfalfa cultivars. Transcriptome analysis showed MsSPL12 modulates numerous drought-responsive genes, with ABA biosynthesis genes closely associated with altered ABA levels in transgenic plants. Biochemical assays verified that MsSPL12 directly activates the expression of MsBCH2 and MsCCD4, key genes in ABA biosynthesis, thereby promoting ABA accumulation. Overexpression of MsBCH2 and MsCCD4 significantly increased ABA content and drought tolerance in alfalfa plants and hairy roots, respectively. This study reveals the regulatory module of MsSPL12-mediated ABA biosynthesis in alfalfa drought tolerance, providing valuable molecular targets and uORF-based strategies for drought-tolerant alfalfa breeding.