MAC5A dual-functionally regulates cold responses at both transcriptional and post-transcriptional levels

INTRODUCTION: Plant cold responses have been extensively studied at the transcriptional level, particularly focusingon conserved transcription factors (TFs) such as C-repeat binding factors (CBFs). However, the post-transcriptional roles of RNA-binding proteins (RBPs) remain largely unexplored, with evolutionarily conserved cold-responsive RBPs yet to be investigated across species. OBJECTIVES: This study aimed to identify conserved cold-responsive RBPs across plant species and investigate how a candidate RBP, MAC5A, regulates cold tolerance. METHODS: Integrative analyses of multi-species comparative genomes and low-temperature transcriptomes identified conserved cold-responsive RBPs. MAC5A loss-of-function andoverexpression lines were examinedto validate its genetic role in cold tolerance. Comparative transcriptomics and CUT&Tag-seq were appliedto determine MAC5A-modulated alternative splicing (AS) and cold-regulated (COR) gene expression, which were further validatedthrough minigene and dual-luciferase assays. RESULTS: We identified multiple conserved cold-responsive RBPs and focused on Arabidopsis MAC5A, a spliceosome component. Cold-induced MAC5A expression was independent of both CBF regulation and ABA signaling. Loss-of-function of MAC5A decreased freezing tolerance, while its overexpression enhanced it. The mac5a mutant exhibited extensive cold-responsive transcriptome alterations, with thousands of genes showing differential AS. Minigene assays confirmed MAC5A modulates splicing of COR genes, including CCA1,PRR9,ABF3 andbHLH112. Additionally, MAC5A regulated expression of other COR genes such as ABI1, COR28, GolS2, RD20, GRP4, DIN10 and NIA1 at the transcriptional level, likely through protein complexes. Notably, limited overlap existed betweenMAC5A-modulated AS genes andtranscriptionally regulated CORgenes, suggesting largely independent regulatory pathways. CONCLUSIONS: MAC5A functions as a dual regulator to orchestrate cold response through both transcriptional and post-transcriptional control of COR genes. The limited overlap between genes regulated by these two layers indicates potential uncoupled regulatory manners, warranting further investigation into their coordination during cold stress.