Biochemical characterization of Selaginella 14-3-3 protein isoforms provides insights into the evolution of their phylogenetic groups

Numerous isoforms of 14-3-3 proteins regulate plant growth, nutrient uptake, flowering, and signaling by interacting with phosphorylated partner proteins. Recently, the two major phylogenetic groups of 14-3-3 isoforms in Arabidopsis thaliana, epsilon and non-epsilon, were found to have distinct biochemical properties, but 14-3-3 proteins in non-model and early-diverging plant lineages remain unexplored. Here, we report a comparative study of 14-3-3 isoforms from Selaginella moellendorffii, a lycophyte representing ancient vascular plants. We show that, like other known 14-3-3s, all Selaginella 14-3-3 isoforms form homodimers and bind phosphopeptides. However, the epsilon-type isoform Sm230088 displays a tendency to monomerize, as recently discovered for Arabidopsis epsilon-type isoforms, but to a lesser extent. Although Sm230088 exhibits lower thermodynamic and proteolytic stability and higher surface hydrophobicity than its non-epsilon counterparts, Sm439395 and Sm229825, Selaginella 14-3-3 isoforms are overall much more stable and form tighter dimers than those in A. thaliana, with notably less pronounced biochemical differences between epsilon and non-epsilon isoforms. This suggests that plant 14-3-3 proteins may have evolved reduced stability over time, while back extrapolation of this trend for each phylogenetic group indicates that, contrary to previous assumptions, plant 14-3-3 proteins likely originated from ancestral non-epsilon, rather than epsilon forms.