Unraveling the evolution of the ATB2 subgroup basic leucine zipper transcription factors in plants and decoding the positive effects of BdibZIP44 and BdibZIP53 on heat stress in Brachypodium distachyon.

In plants, basic region/leucine zipper motif (bZIP) transcription factors (TFs) stand as pivotal regulators in a broad spectrum of developmental mechanisms and adaptive strategies against environmental pressures. However, the ancestral origins and the evolutionary progression of their functional diversity across plant species have yet to be thoroughly illuminated. This study delved into the ATB2 subgroup bZIP homologs, tracing them back to the ancestral charophyte lineage predating land plant emergence, and categorized them into four distinct phylogenetic clusters (Clades A to D). Of particular note, our findings highlighted bZIP44_GBF6 and bZIP53 orthologs as angiosperm-specific innovations, distinguished by the acquisition of novel protein motifs and an intensified regime of purifying selection, reflecting their specialized evolutionary trajectories. Through synteny analysis, we uncovered that whole-genome duplication (WGD) events, post-monocot/dicot split, have played independent yet pivotal roles in shaping the bZIP44_GBF6 and bZIP53 lineages. Furthermore, an assessment of codon usage patterns disclosed a conspicuous bias in monocots favoring G3s, C3s, Gc3s, and GC content, while demonstrating a relative avoidance of T3s, A3s, and Nc usage frequencies. Functionally, we demonstrated that BdibZIP44 and BdibZIP53, localized to the nucleus, possessed the capability to dimerize, both homotypically and heterotypically. These proteins exhibited inducible expression under heat stress conditions in Brachypodium distachyon, implicating them in thermotolerance mechanisms. Overexpression studies reinforced their positive regulatory influence on heat stress resilience by augmenting the enzymatic activity of antioxidants, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), which collectively enhance the clearance of deleterious reactive oxygen species (ROS). Taken together, this research significantly advanced our understanding of the origins and the adaptive evolutionary journey of ATB2 subgroup bZIP homologs in the plant kingdom. Moreover, it elucidated the vital roles of BdibZIP44 and BdibZIP53 in orchestrating a robust defense against high-temperature stress, thereby contributing to the broader discourse on plant adaptation and survival strategies under changing climatic conditions.