RESUMO
The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel. Both channels are highly expressed in Arabidopsis tissues, including root hairs and guard cells that experience rapid turgor changes during root-hair elongation and stomatal movements. Disruption of these two genes, either in single or double mutants, resulted in shorter root hairs and smaller stomatal aperture, with double mutants showing more severe defects, suggesting that these two channels function additively to facilitate anion influx into the vacuole during cell expansion. In addition, dtx35 single mutant showed lower fertility as a result of a defect in pollen-tube growth. Indeed, patch-clamp recording of isolated vacuoles indicated that the inward chloride channel activity across the tonoplast was impaired in the double mutant. Because MATE proteins are widely known transporters of organic compounds, finding MATE members as chloride channels expands the functional definition of this large family of transporters.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Canais de Cloreto/genética , Regulação da Expressão Gênica de Plantas , Membranas Intracelulares/metabolismo , Proteínas de Membrana Transportadoras/genética , Raízes de Plantas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Membranas Intracelulares/ultraestrutura , Potenciais da Membrana/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Pressão Osmótica , Técnicas de Patch-Clamp , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Raízes de Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Nicotiana/genética , Nicotiana/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 through AtPRK5, but not of AtPRK6, in pollen growth were analyzed in tobacco. Herein, AtPRK6 was cloned, and its function was identified. AtPRK6 was expressed specifically in pollen tubes. A yeast two-hybrid screen of AtPRK6 against 14 Arabidopsis Rop guanine nucleotide exchange factors (RopGEFs) showed that AtPRK6 interacted with AtRopGEF8 and AtRopGEF12. These interactions were confirmed in Arabidopsis mesophyll protoplasts. The interactions between AtPRK6 and AtRopGEF8/12 were mediated by the C-termini of AtRopGEF8/12 and by the juxtamembrane and kinase domain of AtPRK6, but were not dependent on the kinase activity. In addition, transient overexpression of AtPRK6::GFP in Arabidopsis protoplasts revealed that AtPRK6 was localized to the plasma membrane. Tobacco pollen tubes overexpressing AtPRK6 exhibited shorter tubes with enlarged tips. This depolarized tube growth required the kinase domain of AtPRK6 and was not dependent on kinase activity. Taken together, the results show that AtPRK6, through its juxtamembrane and kinase domains (KD), interacts with AtRopGEF8/12 and plays crucial roles in polarized growth of pollen tubes.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/química , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Nicotiana/genéticaRESUMO
Abscisic acid (ABA) regulates numerous physiological and developmental processes in plants. Recent studies identify intracellular ABA receptors, implicating the transport of ABA across cell membranes as crucial for ABA sensing and response. Here, we report that a DTX/Multidrug and Toxic Compound Extrusion (MATE) family member in Arabidopsis thaliana, AtDTX50, functions as an ABA efflux transporter. When expressed heterologously in both an Escherichia coli strain and Xenopus oocyte cells, AtDTX50 was found to facilitate ABA efflux. Furthermore, dtx50 mutant mesophyll cells preloaded with ABA released less ABA compared with the wild-type (WT). The AtDTX50 gene was expressed mainly in the vascular tissues and guard cells and its expression was strongly up-regulated by exogenous ABA. The AtDTX50::GFP fusion protein was localized predominantly to the plasma membrane. The dtx50 mutant plants were observed to be more sensitive to ABA in growth inhibition. In addition, compared with the WT, dtx50 mutant plants were more tolerant to drought with lower stomatal conductance, consistent with its function as an ABA efflux carrier in guard cells.