In this doctoral thesis we have used two complementary approaches to study the molecular basis of A. thaliana pH homeostasis. On the one hand, we have performed a genetic screening using acetic acid to look for intracellular acidification tolerant mutants. On the other hand, we have studied acetic acid transcriptional response in Arabidopsis thaliana.
        The genetic screening of an activation tagging mutant seed collection has led to the isolation of the mutant wat1-1D (“weak acid tolerant1-allele1 dominant), which is able to germinate and establish its seedlings faster than the wild type in the presence of acetic acid. Wat1-1D is a negative dominant mutant, whose mutation is localized in the At3g55480 locus that codifies for a ?3 adaptin. This protein is involved in protein transport to the tonoplast, and its loss of function can cause vacuolar transporters mislocalization. Cotyledon emergence experiments showed that wat1-1D mutant is more tolerant to weak acids, ABA and osmotic stress (NaCl, mannitol), but it’s more sensitive to toxic cations such as lithium and norspermidine. In later stages, the mutant is able to maintain a higher pH during an acetic acid perfusion experiment. The analysis of the different mechanisms of pH homeostasis regulation indicated that in the presence of acetic acid, the mutant extrudes more protons to the extracellular media than the wild type, but this doesn’t happen in normal conditions. This proton extrusion doesn’t induce plasma membrane hyperpolarization, because in the presence of acetic acid, the mutant shows enhaced rubidium uptake (potassium analogous). The lithium sensitivity and the sodium tolerance observed during the first physiological stages, is maintained in adult plants irrigated with these toxic cations. Sodium, lithium and rubidium uptake (short-time treatments) are similar in the wild type and the wat1-1D mutant. However, after a two day treatment, the mutant accumulates more potassium in roots and shoots, and less sodium and lithium in the roots.
        Based in these results, we postulate a model in which the mutation in the ?3 adaptin would cause a partial mislocalization of a vacuolar potassium transporter to the plasma membrane. The lack of this transporter in the vacuole, would impair potassium exit, causing tonoplast hyperpolarization that would reduce lithium entry into the vacuole. This decrease in vacuolar lithium uptake would reduce the global lithium accumulation, but it would accumulate in the cytosol where it’s more toxic. Futhermore, the tonoplast hyperpolarization would reduce vacuolar H+-ATPase activity, and this would indirectly affect NHX antiporters, reducing also sodium entry into the vacuole. Finally, under acetic acid stress, the potassium transporter (now localized in the plasma membrane), would become more active, increasing potassium uptake. This would depolarize the plasma membrane, and would favour the proton extrusion observed in the mutant under these conditions.
        The transcriptional analysis that has been done in this doctoral thesis shows that acetic acid treatment induces several genes involved in abiotic stress (heat, high light, oxidative stress) responses. The comparison between the genes induced by these stresses and those induced by acetic acid, shows that heat stress is the most similar to acetic acid stress. In fact, there are several HSF (Heat Shock Factors) and HSP (Heat Shock Proteins) among the most induced genes. This result suggests that acetic acid stress causes protein denaturation. Futhermore, many nutrient uptake symporters (P, S) are induced in the presence of acetic acid. This probably indicates that intracellular acidification impairs nutrient uptake through proton symporters.