Elucidating the ancestral role of Class I HD-Zip transcription factors in land plants.

Class I homeodomain-leucine zipper (C1HDZ) transcription factors regulate diverse developmental and stress-related processes in land plants. While extensively studied in angiosperms, their functions in other lineages are poorly understood. In the liverwort Marchantia polymorpha, the sole C1HDZ orthologue (MpC1HDZ) controls herbivore defense by regulating oil body cell formation. This suggests that the function of C1HDZ genes may have diverged during the evolution of land plants. Here, we further investigated the function of MpC1HDZ in M. polymorpha and its integration into Arabidopsis (Arabidopsis thaliana) regulatory networks. Mpc1hdz mutants were more susceptible to herbivory by Spodoptera frugiperda caterpillars but displayed greater resistance to the oomycete Phytophthora palmivora, indicating that MpC1HDZ-mediated defenses extend to other herbivores but not oomycete pathogens. Loss-of-function Mpc1hdz mutants exhibited developmental alterations, including shortened gemma cups, diminished air pore density, and absence of surface papillae, in addition to the loss of oil bodies, highlighting a broader role for MpC1HDZ in epidermal cell differentiation. Heterologous expression of MpC1HDZ in the Arabidopsis athb13-2 mutant resulted in a smaller rosette diameter, greater leaf number and serration, and shortened inflorescence height; it also restored drought tolerance and improved the forage quality of the mutant tissues. These findings demonstrate that MpC1HDZ can be functionally integrated into endogenous HD-ZIP I regulatory networks, modulating C1HDZ-associated responses. Together, these results support an ancestral developmental role for C1HDZ genes, with abiotic stress functions emerging later in angiosperm evolution, enabled by conserved protein features that facilitate regulatory network integration.