Characterization and Role of AP2/EREBP Genes with Decreasing Expression During Leaf Development in 84K Poplar

The 84K poplar (Populus alba × Populus glandulosa) is a fast-growing hybrid poplar that was introduced from South Korea by the Chinese Academy of Forestry in 1984. To gain deeper insight into the regulatory mechanisms of leaf development in 84K poplar, we performed bulk RNA sequencing and found that numerous members of the AP2/EREBP family exhibited expression changes, suggesting their crucial roles in leaf development. The AP2/EREBP transcription factor family is one of the largest and most conserved gene families in plants. These genes play a crucial role in plant growth, development, and stress responses. In this study, we identified and analyzed 400 AP2/EREBP genes through transcriptome analysis, excluding genes with missing values (NAs) or FPKM < 1, and selected 76 genes based on their expression patterns at different stages of leaf development. The 76 genes were classified into three subfamilies based on phylogenetic analysis and structural domain characteristics: the RAV subfamily, the ERF subfamily, and the AP2 subfamily. Each subfamily shares similar gene structures and motifs while also exhibiting distinct differences. Segmental duplication events may have contributed to the evolution of this gene family. Most of the promoter cis-acting elements are related to light responses, with fewer elements associated with palisade tissues and hormones. Eight genes, selected for their gradually decreasing expression during leaf development, were validated through RT-PCR experiments. Among them, five genes-Pop_G10G022861, Pop_A01G003858, Pop_A01G081120, Pop_A01G074798, and Pop_A07G010900-exhibited a decreasing trend in expression across the three stages of leaf development. Subcellular localization analysis indicated that Pop_A01G003858 and Pop_G11G077730, two randomly selected genes from the eight AP2/EREBP members validated by RT-PCR, are localized in the nucleus. In conclusion, these findings provide valuable insights into the evolutionary relationships of the 73 AP2/EREBP family members in 84K poplar leaves and lay a foundation for future studies on leaf development.