The coevolution process has shaped the diversity and complexity of life on Earth. Coevolution involves the interaction between two entities in a highly specific process because selective pressures in one of the entities drive the evolution of the other. Moreover, coevolution is reciprocal and simultaneous, as evolution occurs in both entities at the same time. At the molecular level, molecular coevolution occurs in systems where molecules interact closely (e.g., enzymes and their substrates, proteins and their binding partners, transcription factors and their specific binding motifs on DNA, etc.). Many factors play a pivotal role in shaping molecular coevolution, such as drift intensities, selection, and mutation rates. Intramolecular coevolution occurs between sites within a single molecule, while intermolecular coevolution takes place between sites of two interacting molecules. Identifying coevolving sites is crucial for understanding protein-protein interactions, drug resistance, the evolutionary arms races between hosts and pathogens, and for enhancing molecular activity.

Plant response mechanisms to salinity stress are diverse and complex. Among them, those involved in ion homeostasis and compartmentalisation play a key role in isolating and eliminating toxic ions. The main strategies are the expression of Salt Overly Sensitive (SOS) genes, the High-Affinity Potassium Transporters (HKTs), the Na⁺/H⁺ Antiporter (NHX), and the proton pumps. The aim of this work is, first, to identify coevolving amino acids within these proteins and, second, to find if there exist any coevolving sites between them. We used CAPS, a software to identify co-evolution between amino acid sites by measuring the correlated evolutionary variation at those sites, which further removes the phylogenetic and stochastic dependencies between sites. The software uses a reference sequence for which the 3D protein structure is available. NHX1, SOS1, SOS2, and HKT1 were analysed, and we found pairs of sites under strong intramolecular co-evolution as well as intermolecular interactions.