Caracterización funcional de las proteínas mtv9 y mtv11 y su implicación en el tráfico vacuolar

Resumen

This doctoral thesis adress the characterization of MTV genes that are involved in vacuolar trafficking in Arabidopsis. The vacuole is an essential organelle for adaptative strategies of plants and has a great agronomic and biotechnological importance for its storage capacity of essential proteins for human nutrition or of recombinant proteins for biotechnological purposes. In this thesis, two mutants modified in trafficking to vacuole (mtv mutants) have been studied and point mutations in the MTV9 / At1g24560 and MTV11 / At4g29380 genes have been identified as the responsible for the defects in vacuolar trafficking. The MTV9 gene is plant-specific and encodes a protein that is located in the pre-vacolar compartment, possibly directed to that compartment by the palmitoylation of a cysteine present at its C-terminal end. The overexpression of MTV9 provokes the aggregation of the pre-vacuolar compartments and the delocalization of SNARE proteins, interfering with the trafficking of proteins to vacuoles but not with the secretion of plasma membrane proteins. These results indicate that MTV9 is essential for the trafficking to vacuoles and could be involved in anchoring processes of vesicles or organelles with the prevacuolar compartment to mediate their fusion. The MTV11 gene encodes for the ortholog of the VPS15 protein, which is part of a phosphatidylinositol-3-kinase (PI3K) complex, whose activity is required for all vacuolar related trafficking routes in yeasts and animals. The MTV11 gene is essential in plants and its disruption causes lethality in pollen. The mutant mtv11-1 isolated in this thesis is a hypomorphic allele that has allowed to study the role of this gene Arabidopsis growth and development. By using the biomarker 2xFYVE we have obtained evidence that the synthesis of phosphatidylinositol-3-phosphate is significantly decreased in the mutant, indicating that the mutant allele reduces the activity of the PI3K complex. The mutant mtv11-1 has affected the trafficking of storage proteins in seeds reservoirs and the recycling of plasma membrane proteins. These defects cause a decrease in growth and alterations in filotaxia under normal growth conditions. Moreover, different assays to study resistance to abiotic stresses performed in the mtv11-1 mutant, have shown that mtv11-1 plants are more tolerant to high concentrations of arsenate, a compound that is sequestered in vacuoles once it is reduced to arsenite in the cytosol. This discovery may have important implications for the development of improved plants in phytoremediation of soils contaminated with arsenic.