Biochemical signatures strongly demarcate phylogenetic groups of plant 14‐3‐3 isoforms

Interaction of dimeric 14-3-3 proteins with phosphotargets regulates various physiological processes in plants, from flowering to transpiration and salt tolerance. Several genes express distinct 14-3-3 "isoforms," particularly numerous in plants, but these are unevenly studied even in model species. Here we systematically investigated twelve 14-3-3 isoforms from Arabidopsis thaliana. While all these proteins can homodimerize, four isoforms representing a supposedly more ancestral, epsilon phylogenetic group (iota, mu, omicron, epsilon), but not their eight non-epsilon counterparts (omega, phi, chi, psi, upsilon, nu, kappa, lambda), exhibit concentration-dependent monomerization, and pronounced surface hydrophobicity at physiologically relevant protein concentrations and under crowding conditions typical for the cell. We show that dramatically lowered thermodynamic stabilities entail aggregation of the epsilon group isoforms at near-physiological temperatures and accelerate their proteolytic degradation in vitro and in plant cell lysates. Mutations in 14-3-3 iota, inspired by structural analysis, helped us rescue non-epsilon behavior and pinpoint key positions responsible for the epsilon/non-epsilon demarcation. Combining two major demarcating positions (namely, 27th and 51st in omega) and differences in biochemical properties, we developed an epsilon/non-epsilon demarcation criterion that classified 89% of available 14-3-3 sequences from Dicots, Monocots, Gymnosperms, Ferns, and Lycophytes with 99.7% accuracy, and reliably predicted biochemical properties of a given 14-3-3 isoform, which we experimentally verified for distant 14-3-3 isoforms from Selaginella moellendorffii. The proven occurrence of isoforms of both groups in primitive plants refines the traditional phylogenetic, solely sequence-based analysis and provides intriguing insights into the evolutionary history of the epsilon phylogenetic group.