Evolutionary Diversification and Adaptive Evolution Analysis of the Plant HD-Zip IV Subfamily

<b>Background/Objectives:</b> The terrestrialization of plants has been a significant driver of plant evolution over hundreds of millions of years, and epidermal development plays a crucial role in adapting to terrestrial environments. It is therefore of great interest that the HD-Zip IV subfamily serves as a key regulator of epidermal and cuticle development in plants. However, research on their expansion trajectory, adaptive evolution, and functional divergence remains scarce. <b>Methods:</b> We conducted a functional divergence and adaptive evolution analysis in the plant HD-Zip IV subfamily and confirmed the functional differentiation caused by positive selection site mutations through binding affinity prediction analysis and EMSA experiments. <b>Results</b>: Our findings revealed that the HD-Zip IV subfamily has diversified into five distinct branches, with progressive expansion throughout plant evolution. These variations in the HD (homeodomain) and START drive the functional differentiation among the evolutionary branch, in particular, a distinct leucine-rich motif in HD and lipid-binding pockets. Furthermore, we identified several amino acid sites within the START domain that have been under selective pressure during plant evolution, as well as convergent evolutionary sites shared between early land plants and seed plants. <b>Conclusions</b>: Our findings show that the HD-Zip IV subfamily experienced a significant expansion in gene number and diversification of evolutionary branch from streptophyte algae to seed plants. During plant evolution, genomic duplication events and variations in the HD and START have contributed to its expansion in gene number and diversification, respectively. We suggest that the balanced coexistence of functional robustness and relaxed constraints in the HD-Zip IV subfamily may have underpinned its successful response to the challenges of land environment.