Single Amino Acid Divergence Drives Functional Specialisation of Homologous GRFs in Populus.

Gene duplication with sub- and/or neo-functionalization is a key pathway in plant adaptive evolution. The duplication-degeneration-complementation (DDC) model explains sub-functionalization of paralogs, yet the relative contributions of coding sequence versus cis-regulatory changes remain contested. In this study, we show that a single amino acid substitution (Met30/Ile30) in the WRC domain drives sub-functionalization of poplar paralogs PagGRF29 and PagGRF10, demonstrating a key role for coding changes. Met30 confers high DNA-binding capacity and represents the ancestral state, whereas Ile30 severely impairs DNA binding. Evolutionary analysis reveals residue 30 as a conserved tuning site, with the degenerative Ile30 variant emerging during plant terrestrialization. Despite impaired DNA binding, PagGRF10 physically interacts with PagGRF29 to form a complementary module: PagGRF29 alone enhances drought tolerance at growth expense, while the PagGRF29-PagGRF10 complex promotes biomass accumulation with reduced stress resilience. Thus, the Met30/Ile30 switch drives the resolution of duplicated genes through DNA-binding-based functional specialisation and cooperative regulatory module formation, orchestrating the growth-stress trade-off. This pattern represents an extended manifestation of the DDC framework, combining asymmetric sub-functionalization with interaction-mediated functional innovation. Our study demonstrates how a single-amino-acid substitution can drive paralog specialisation, offering mechanistic insight into the evolutionary fates of duplicated genes in plants.