Structure and regulation of NHX exchangers in the uptake of potassium into the vacuoles of arabidopsis thaliana.

Abstract

Salinity stress is one of the main causes of crop loss today in semi-arid and irrigated agricultural land. Due to the sessile nature of the plants, they have developed mechanisms that allow them to adapt, tolerate and survive to these conditions, and that include preventing the toxicity produced by the sodium and chloride ions, maintaining potassium homeostasis and optimizing the use of resources. How these tolerance mechanisms are activated and how they work is of interest, not only to increase crop production, but also to optimize their use in soils non suitable for agriculture in the poorest regions of the world in order to ensure global food security. Maintaining the cytosolic K+/Na+ ratio in cells is critical for preserving the functionality of cell components, and thus one of the main mechanism leading to salt stress tolerance. In Arabidopsis, the NHX family of K+,Na+/H+ antiporters comprises eight members (AtNHX1-8) which are highly related to this tolerance mechanism. They can be divided in vacuolar (AtNHX1-4), endosomal (AtNHX5- 6) or plasma membrane (AtNHX7-8) exchangers according to their location. These proteins belong to the highly conserved cation/proton antiporters (CPA) superfamily, which is present in all kingdoms of nature. Based in the knowledge gained from microbial counterparts, we studied the structure-function relationship of AtNHX1. These analyses allowed us to generate a topological and tridimensional model, as well as to identify conserved amino acids which, by model-guided mutagenesis, we probed essential for the activity of the protein. We also studied the regulation of AtNHX1 by pH and by the interaction with CML18. The calmodulin-binding domain was also characterized and by using model-guided techniques and mutagenesis we generated a model of the interaction mechanism and the implications on the regulation of AtNHX1 activity. In this model, the calmodulin binding domain of AtNHX1 integrates a cis-regulation by cytosolic pH and the trans-regulation by CML18 and calcium. CML18 binding stimulates K+/H+ antiport at the tonoplast as long as the cytosolic pH is not exceedingly acidic, in which case protonation of H499 promotes the formation of a salt bridge with D506, thereby hindering the binding of CML18 to restrain further ion exchange and the release of protons into the cytosol. Plants subjected to saline stress activate a response mechanism that involves the activity of a large number of membrane transporters. Among the main secondary messengers that mediate these processes there are the pH fluctuations, as a consequence of H+-linked ion fluxes, and Ca2+ transients. Tracking these changes in time and space in roots and seedlings of Arabidopsis lines expressing the genetically encoded pHGFP sensors and real-time imaging techniques, we were able to record cytosolic pH changes generated after NaCl treatments in the vicinity of the plasma membrane and tonoplast, and in the cytoplasm. These maps my shed light into the mechanism of stress perception and tolerance. As a proof of concept, we carried out the experiments with the sos3 mutant of Arabidopsis lines that are impaired in mounting the salt-stress response aiming to reduce Na+ toxicity by the SOS pathway. Finally, AtNHX3 and AtNHX4 are two members of the Arabidopsis NHX that have been largely overlooked. With the aim of understanding their implication plant development and in the salt tolerance of Arabidopsis thaliana, single and double nhx3 and nhx4 mutants were generated and their phenotype analyzed under different conditions. We determined that these proteins have their own functional niche, although they can also contribute to salt stress in a small proportion. AtNHX3 seems to be related to regulate the vacuolar pH in petals in the process of flower opening.