Pb can damage the physiological function of human organs by entering the human body via food-chain enrichment. Revealing the mechanisms of maize tolerance to Pb is critical for preventing this. In this study, a Pb-tolerant maize inbred line, 178, was used to analyse transcription factors (TFs) expressed under Pb stress based on RNA sequencing data. A total of 464 genes expressed in control check (CK) or Pb treatment samples were annotated as TFs. Among them, 262 differentially expressed transcription factors (DETs) were identified that responded to Pb treatment. Furthermore, the DETs were classified into 4 classes according to their expression patterns, and 17, 12 and 2 DETs were significantly annotated to plant hormone signal transduction, basal transcription factors and base excision repair, respectively. Seventeen DETs were found to participate in the plant hormone signal transduction pathway, where basic leucine zippers (bZIPs) were the most significantly enriched TFs, with 12 members involved. We further obtained 5 Arabidopsis transfer DNA (T-DNA) mutants for 6 of the maize bZIPs, among which the mutants atbzip20 and atbzip47, representing ZmbZIP54 and ZmbZIP107, showed obviously inhibited growth of roots and above-ground parts, compared with wild type. Five highly Pb-tolerant and 5 highly Pb-sensitive in maize lines were subjected to DNA polymorphism and expression level analysis of ZmbZIP54 and ZmbZIP107. The results suggested that differences in bZIPs expression partially accounted for the differences in Pb-tolerance among the maize lines. Our results contribute to the understanding of the molecular regulation mechanisms of TFs in maize under Pb stress.

Among eight Arabidopsis CaM-Binding Protein (CBP) 60 family members, AtCBP60g and AtSARD1 are partially functionally redundant, major positive immune regulators while AtCBP60a is a negative immune regulator. Phylogenetic analysis of CBP60 protein sequences of 247 diverse land plant species indicated that the CBP60a, CBP60g, and SARD1 immune regulator subfamilies diversified around the time of Angiosperm divergence. The immune regulator subfamilies, especially the CBP60g subfamily, have been evolving very fast, suggesting strong selection pressure from CBP60-targeting pathogen effectors. We closely examined this fast evolution of the immune regulator subfamilies using the subfamily protein sequences from 12 diverse Core Eudicot species. The fast evolution was caused by both high proportions of polymorphic sites and high evolution rates per polymorphic site, compared to the genomic norm. We developed an analytical platform for physical-chemical characteristics of amino acids, called Protein Evolution Analysis in Euclidean Space (PEAES). Using the pairwise distance rank on PEAES (PEAES-PDR), we detected signatures of significant coevolutionary interactions across the immune regulator subfamilies within the CBP60-conserved domain. The patterns of detected coevolutionary interactions were consistent with hypothetical coevolutionary mechanisms that protect the positive immune regulator function from targeting by pathogen effectors. The coevolutionary interactions across the subfamilies were largely specific to each species lineage, suggesting that the coevolution occurred through species lineage-specific histories of variable pathogen pressure. Thus, fast coevolution of the subfamilies with overlapping or opposing functions appears crucial to maintain resilience of the CBP60 immune regulator module against fast-evolving pathogen effectors.