Multi-dimensional demarcation of phylogenetic groups of plant 14-3-3 isoforms using biochemical signatures

Interaction of dimeric 14-3-3 proteins with numerous 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 comparative studies of all 14-3-3 isoforms for a given organism have not been undertaken. Here we systematically investigated twelve 14-3-3 isoforms from the model plant Arabidopsis thaliana, uniformly capable of homodimerization at high protein concentration. We unexpectedly discovered that, at physiological protein concentrations, four isoforms representing a seemingly more ancestral, epsilon phylogenetic group (iota, mu, omicron, epsilon) demonstrate an outstanding monomerization propensity and enhanced surface hydrophobicity, which is uncharacteristic for eight non-epsilon isoforms (omega, phi, chi, psi, upsilon, nu, kappa, lambda). Further analysis revealed that dramatically lowered thermodynamic stabilities entail aggregation of the epsilon-group isoforms at near-physiological temperatures and provoke their proteolytic degradation. Structure-inspired single mutations in 14-3-3 iota could rescue non-epsilon behavior, thereby pinpointing key positions responsible for the phylogenetic demarcation. Combining two major demarcating positions (namely, 27th and 51st in omega) and multi-dimensional differences in biochemical properties identified here, we developed a predictor strongly supporting categorization of abundant 14-3-3 isoforms widely across plant groups, from Eudicots to Monocots, Gymnosperms and Lycophytes. In particular, our approach fully recapitulates the phylogenetic epsilon/non-epsilon demarcation in Eudicots and supports the presence of isoforms of both types in more primitive plant groups such as Selaginella, thereby refining solely sequence-based analysis in evolutionarily distant species and providing novel insights into the evolutionary history of the epsilon phylogenetic group. SignificanceDespite over 30 years of research, systematic comparative studies on the regulatory plant 14-3-3 proteins have not been undertaken, making phylogenetic classification of numerous plant 14-3-3 isoforms in different species unreliable. Working on twelve purified Arabidopsis 14-3-3 isoforms, we have discovered a set of biochemical signatures that can be used to robustly and widely categorize epsilon and non-epsilon plant 14-3-3 isoforms, also identifying at least two amino acid positions responsible for such multi-dimensional demarcation.