Integrative multi-omics analysis of three early diverged rosid species reveals an ancient hierarchical cold-responsive regulatory network.

Elucidating regulators, including transcription factors (TFs) and RNA-binding proteins (RBPs), underlying gene transcriptional and post-transcriptional co-regulatory network is key to understand plant cold responses. Previous studies were mainly conducted on single species, and whether the regulators are conserved across different species remains elusive. Here, we selected three species that diverged at the early evolution of rosids (~99-113 million years ago), performed cold-responsive phylotranscriptome experiments, and integrated chromatin immunoprecipitation- and DNA affinity purification-sequencing (ChIP/DAP-seq) analysis to explore cold-responsive regulators and their regulatory networks. First, we detected over 10,000 cold-induced differentially expressed genes (DEGs) and alternative splicing genes (DASGs) in each species. Among the DEGs, a set of TFs and RBPs were conserved in rosid cold response. Compared to TFs, RBPs displayed a delayed cold-responsive pattern, implying a hierarchical regulation of DEGs and DASGs. By integrating DEGs and DASGs, we identified 259 overlapping DE-DASG orthogroups (closely-related homologs) that were cold-regulated at both transcriptional and post-transcriptional levels in all three studied species. Notably, pathway analysis on each of the DEGs, DASGs, and DE-DASGs in the three species showed a common enrichment connected to the circadian rhythm. Evidently, 226 cold-responsive genes were directly targeted by at least two circadian rhythm components (CCA1, LHY, RV4, RVE7, and RVE8). Finally, we revealed an ancient hierarchy of cold-responsive regulatory networks at transcriptional and post-transcriptional levels launched by circadian components in rosids. Altogether, this study sheds light on conserved regulators underlying cold-responsive regulatory networks across rosid species, despite a long evolutionary history after their divergence.