Evolutionary and functional analyses in angiosperms reveal a conserved cold-responsive WRKY33-NAC032 module

Angiosperms have colonized diverse climates, ranging from tropical to temperate and polar regions. While the C-repeat binding factor (CBF)-mediated regulatory network is a well-established mechanism in plant cold responses, the existence of other evolutionarily conserved pathways remains poorly understood. We conducted comparative genomic and transcriptomic analyses of 10 representative angiosperms exposed to cold stress, followed by functional characterization of selected regulatory factors in both the herbaceous model Arabidopsis thaliana and the woody species Betula platyphylla (birch) through genetic and molecular approaches. We identified 22 conserved cold-responsive transcription factor orthogroups (CCRTFOs), including members of the CBF, WRKY, ERF and NAC families. Genetic analysis of the WRKY25/26/33 orthogroup revealed that WRKY25 and WRKY33 confer cold responses via a CBF-independent pathway. Mechanistically, WRKY33 directly regulates the expression of NAC032, a member of another CCRTFO, which we also functionally validated as a positive regulator of cold tolerance. Moreover, cross-species validation confirmed that the birch orthologs, BpWRKY33 and BpNAC032, similarly improve cold tolerance. Collectively, our findings identify a core set of CCRTFOs and demonstrate the evolutionary conservation and functional significance of the WRKY33-NAC032 module in angiosperm cold resistance, highlighting a broader molecular network beyond the canonical CBF pathway.