Evolutionary studies of the basic helix-loop-helix (bHLH) IVc gene family in plants and the role of AtILR3 in Arabidopsis response to ABA stress

The basic helix-loop-helix (bHLH) IVc transcription factors (TFs) play central roles in controlling iron (Fe) homeostasis and biotic stress responses. However, their evolutions and functions in other abiotic stresses are poorly understood. In this study, the IAA-LEUCINE RESISTANT3 (ILR3) homologs were traced roughly back to before the early origin of land plants and divided into six main clades (Clade A-F). Further analysis found that the ILR3 orthologs were angiosperm-specific, suffering from motif-acquisition events and loose purifying selection. Synteny analysis displayed that the whole genome duplications (WGDs) contributed to the establishment of the IRON DEFICIENCY TOLERANT1 (IDT1, also called bHLH34)/bHLH104 lineage prior to the divergence of angiosperms. Sequence analysis revealed that the ILR3 homologs had some novel and conserved motifs except bHLH and leucine zipper (ZIP) domains. Particularly, Arabidopsis thaliana ILR3 (AtILR3) was a nuclear protein and greatly activated by ABA and CdCl2 stresses. Simultaneously, the molecular and genetic analyses suggested that the AtILR3 acted as a positive regulator in the ABA stress response through enhancing the ability of reactive oxygen species (ROS)-scavenging, such as the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). The inductively coupled plasma mass spectrometry (ICP-MS) analyses exhibited that the AtILR3 affected the absorption of nutrient elements, especially iron elements, under ABA stress. Collectively, our findings could shed deep light on the origin and evolution of the plant ILR3s, as well as the functions of the AtILR3 under ABA stress.