Función y localización de los transportadores de plantas SOS1 mediante expresión heteróloga en bacteria y levadura

Abstract

Salinity is one of the main problems related to agricultural production, since it affects the plant growth. Most of crop species are glycophytes, which are sensitive to salinity. That is the reason why there is an interest in studying the defense mechanisms of plants against saline stress. Potassium is an essential ion for plants, while sodium can become toxic at high concentrations, which is what plants face when they grow in saline conditions. Salinity produces a toxic and osmotic effect on plants due to the increase of the entrance and accumulation of sodium, altering the physiological K+/Na+ ratio. Plants have two mechanisms to reduce Na+ cytoplasmic levels: they could compartmentalize it into cellular organelles such as vacuoles, or they could extrude it outside; in this last mechanism SOS1 transporters highlight. SOS1 proteins are very important in the defense of plants against salinity. They are Na+/H+ antiporters located in the plasma membrane of epidermal root cells, where the extrude Na+ out of plants, they are also located in xylem parenchyma cells loading Na+ to the xylem. In recent work done in the laboratory, it has been compared by heterologous expression in yeast mutants the functioning of SOS1 of a halophyte species, Eutrema salsugineum, with SOS1 of the glycophyte Arabidopsis thaliana. Results showed differences in their functioning as ionic transporters depending on the pH of the medium. In order to know whether differences in SOS1 functions of the two species, were associated with a difference in subcellular location, SOS1-GFP fusion proteins were constructed in this work, using the new ligation method named NEBuilder HiFi DNA Assembly and expressed in yeast mutants. At pH 5.5 and 6.5 both proteins were detected mainly in plasma membrane. However, at pH 7.5 A. thaliana SOS1 showed a preferential location in plasma membrane, whereas E. salsugineum SOS1 was observed at the plasma membrane as well as in internal cellular membranes. Although the exact intracellular location could not be determined, the difference shown with respect to A. thaliana could explain the difference in the function. On the other hand, in laboratory assays, it has been observed that silenced plants in E. salsugineum SOS1 gene have shown an increase of K+ content in the aerial part. These results suggested a possible role of SOS1 as a Na+/K+ antiporter. To find out whether EsSOS1 and AtSOS1 proteins can transport K+, they were expressed in the Escherichia coli TKW4240 mutant which is defective in its endogenous K+ uptake systems. Drop test complementation assays showed that the expression of SOS1 proteins recovered the ability of the mutant to grow at low K+ concentrations while there was no growth when SpSod2 was expressed, which is a known antiporter that works in a specific way only transporting Na+ against the H+ gradient. These results suggest that SOS1 transporters can transport K+. In this work, different assays have been conducted to study the function of transporters and their GFP fusions. Yeast assays have shown that constructions obtained were functional. In addition, it has been demonstrated that SOS1 has to be regulated by the SOS2- SOS3 complex in order to improve its function. Finally, the well-known function of SOS1 as Na+ transporters was also studied by heterologous expression in yeast and bacterial mutants. While in yeast a clear complementation of the sensitivity to grow in saline conditions was observed, in the bacterial mutants no conclusive results were obtained, so they should be studied in more detail in the future.