Time-course analysis and transcriptomic identification of a groupⅢ ERF CmTINY2 involved in waterlogging tolerance in chrysanthemums

‘Hangju’ is a variety of Chrysanthemum morifolium Ramat with both aesthetic and medicinal value, cultivated as traditional Chinese medicine for four centuries. The cultivation of ‘Hangju’ faces a significant challenge due to waterlogging stress, resulting in a considerable decrease in crop yield. Nevertheless, there is a scarcity of studies exploring the underlying mechanisms of waterlogging tolerance in ‘Hangju’. This investigation focused on comparing the characteristics of the waterlogging-tolerant variety EWT and the waterlogging-sensitive variety CK of ‘Hangju’. The study revealed that EWT exhibited a more advanced aeration tissue structure and demonstrated rapid development of adventitious roots following waterlogging. Through a time-course transcriptome analysis, it was observed that EWT could swiftly adjust the expression of genes involved in energy metabolism signaling pathways to acclimate to the waterlogged environment. WGCNA analysis identified INTEGRASE-TYPE DNA-BINDING PROTEIN (CmTINY2) as a key factor in regulating waterlogging tolerance in EWT. CmTINY2, a transcription factor belonging to the ethylene responsive factor (ERF) subfamily III, operated within the nucleus and activated downstream gene expression. Its role in enhancing waterlogging tolerance might be linked to the control of stomatal aperture. In summary, this research has elucidated that the waterlogging tolerance exhibited by EWT chrysanthemum is a result of a combination of morphological structure and molecular regulatory mechanisms. Furthermore, the discovery of a waterlogging-tolerant gene from the ERF subfamily III has broadened our understanding of the role of ERF genes in waterlogging signaling pathways. Root samples of EWT and CK were collected immediately at 0, 3, 12, 24 h after waterlogging and 24 h after waterlogging with 3 h of reoxygenation (24_3h). Each sample contained four biological replicates. Total RNA was extracted using the Tiangen RNA Extraction Kit with the RNase-Free DNase Set (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions. Libraries for mRNA-seq were constructed and sequenced by Novogene (Beijing). Briefly, RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). mRNAs were isolated from total RNAs by poly(A) selection, fragmented into short fragments, and converted to cDNAs. cDNAs were ligated to adapters and the suitable fragments were selected for PCR amplification as templates. All the RNA-seq libraries were pair-end sequenced on an Illumina Novaseq platform.