Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/20237
Título: Electrophysiologic and molecular characterization of membrane anionic transporters in pollen tubes
Autor: Dias, Pedro Nuno Resende
Orientador: Feijó, José A., 1962-
Silva, Jorge Miguel Luz Marques da, 1965-
Palavras-chave: Arabidopsis thaliana
Membrana plasmática
Teses de doutoramento - 2015
Data de Defesa: 2015
Resumo: The pollen tube is a remarkable cell, playing a fundamental role in the life cycle of higher plants. Some of its characteristics have made it a preferred choice as a model plant system to study apical cell growth and polarization. Apical cell growth and polarization are entangled in pollen tube, and this has been shown to be linked to many extracellular fluxes and internal gradients. Over the years, evidence linking each of these ions, either by their extracellular fluxes or by internal gradients to the polarization and apical growth processes have highlighted their importance and how closely related these phenomena are. Disrupting any of these ionic fluxes invariably leads to pollen tube growth arrest, burst or failure to fertilize the ovules. Still, despite the wealth of knowledge acquired, there is still a large gap in identifying all the genes involved in the ion transport in pollen plasma membrane. A number of channels and pumps have been positively identified over the years, including several cation channels and a number of pumps as well. However, all attempts to identify the molecules responsible for the anion transport have been, so far, unsuccessful. Transcriptomic studies have identified several candidate anionic channels genes that are presented in pollen. Some of them, as the case of the CLC-c, that is highly expressed in pollen, were checked but failed to provide any conclusive result, leaving open the question as to which genes mediate the observed anionic fluxes and gradients. Some of the other candidate genes include promising genes, as the SLAC1 homologues, responsible for the anion fluxes in the guard cell. Another promising candidate is the recently identified TMEM16A homologue, a Ca2+-activated Cl- channel (CaCC) with only one copy in the Arabidopsis genome. Of particular interest is the electrophysiological profile of this channel, that mimic most of the properties that had been previously observed in plant pollen anion currents by means of patch clamp experiments. The question remains about the molecular nature of the anionic transporters in pollen plasma membrane. To answer this, a T-DNA insertion mutant for this particular gene was genotyped and extensively characterized in this thesis. Previous work done in our lab had identified anionic currents in both Arabidopsis thaliana and Lilium longiflorum pollen protoplasts, and linked them to [Ca2+]in regulation. Still, a number of differences from some of the expected values on those currents led to the idea that some other ion could be transported alongside anions under the experimental conditions. The hypothesis is that the observed discrepancies in expected behavior would be caused by pH and H+. To address this and extend our understanding on the nature of the anionic currents, an extensive characterization of the anionic currents was performed. Preliminary results obtained with Lilium longiflorum supported this hypothesis, evidencing a strong regulation by extracellular pH. Focusing on Arabidopsis thaliana pollen to take advantage of the mutant lines available, a strong regulatory effect of extracellular pH was also observed, although with different properties then in Lilium. Under increasing extracellular pH the anionic currents increased dramatically, their conductances changed, the strong outward rectification that characterized them was lost and the current reversal potentials moves toward the expected values for Cl- and H+. These results strongly support the hypothesis that H+ are also being transported along with Cl-, explaining the previously observed discrepancies of the anionic currents, and suggesting the presence of a co-transport system transporting H+ and Cl-. While performing the same experiences in the cacc mutant line a remarkable difference was observed. In the cacc, there is no response to external pH: currents were not largely affected, conductances had slight changes but rectification was unaffected, and the reversal potential did not follow the changing H+ gradient. These results point out to the fact that the CaCC gene is not only a transporter responsible for anion transport in pollen plasma membrane, but specifically an anion/H+ co-transporter. This result is, to our knowledge, the first positive molecular identification of an anion transporter in the plasma membrane of pollen. Even so, the fact remains, that this mutant line shows no obvious phenotypes in terms of reproduction, and most of the differences found were linked to pH. One notable exception is the impact of this mutation on the rundown of the currents. In the mutant the length of rundown is extended, compared to wild type, despite both having a similar percentual loss in current amplitude. This is an indication that the CaCC is also competing for whatever molecule or process is regulating the rundown of the currents. When the anionic concentration was modified both wild type and mutant line evidenced similar behaviors, suggesting that the overall population of anionic channel still conducted anions in a similar way, even in the absence of the CaCC co-transporter. This is not unexpected, given the tight control these processes are subject to, a certain level of redundancy and compensation to overcome the lack of just one transporter is expected. This is perhaps the main reason why it has been so challenging to identify them in the first place. Still, these experiments further validated the fact that Cl- is not the only ion being transported under our experimental conditions, as evidenced by the reversal potential shifts with changing external [Cl-]. Taken together these results with the reversal shifts when pH was changed, we can propose a stoichiometry of 2:1 for the CaCC co-transporter. Furthermore, by substituting the extracellular bath solution [Cl-] for [NO3-] another important result emerged. In wild type, there is no difference, confirming what had been published before, that the anionic channels in the plasma membrane of pollen are equally permeable to Cl- and NO3-, while in the cacc mutant there is a clear difference between the anionic currents under high [Cl-] or high [NO3-]. These results suggest that the CaCC gene should be highly selective for Cl-, and not for NO3-. This would make the CaCC a specific Cl-/H+ co-transporter. These results would make the CaCC present in Arabidopsis pollen to function rather differently than its orthologues in animals. This would not be entirely novel, as many other putative anion channels in plants have recently been show to function not as anion channels, but as co-transporters instead. In fact, our knowledge of anion channels structure and function is still evolving, as many discoveries have highlighted that anion channels to be substantially different from the better studied cation channels, possessing unique properties and behaviors. To further compliment these results the internal pH was also changed, to a more acidic pH, similar to what would be found in the tip of the growing pollen tube. These experiments evidenced quite a few interesting results, revealing a complex regulation by internal pH on the anionic channels and a lack of response in the cacc mutant comparable to that of wild type. With the identification and electrophysiological characterization of an anionic transporter in pollen plasma membrane, we tried to find further evidences of the role of this gene in plant development. A competition assay was performed and while the results indicated a potentially seed set phenotype for the cacc mutant when selfed or reciprocally crossed for the antibiotic resistance, the sample size so far analyzed did not show statistically significant differences. Still, the expected role of the CaCC gene in the overall pollen tube development appears to be limited in scope, as its impact may be masked and compensated by other channels or transporters in circumstances to be determined. As a proof of principle approach, we integrated another electrophysiological technique, the vibrating probe, with the power of the patch clamp technique. This approach would allow us in the future to have a more overarching scope on the characterization of potential new channels. For this, a de novo characterization of the probes dynamic efficiency was made, and a novel species was used to test it. Nicotiana tabacum ionic fluxes were characterized and compared to the well studied Lilium longiflorum. They both evidenced the same spatial pattern in terms of each ion flux spatial distribution across the pollen tube plasma membrane, but with altered amplitudes and temporal patterns. Furthermore, using an altered protocol for data acquisition it was possible to determine that the temporal components of the oscillations observed at the tip also have a spatial distribution that could be used to fine tune the precise location or role of specific genes in future approach. Overall, the anionic currents of Arabidopsis thaliana were extensively characterized. This allowed us to characterize the regulatory effect of pH in these currents, to identify one gene, as an anion/H+ co-transporter, and extensively characterize its mutant and its absence of pH response. This was the first time a gene was positively identified as a plasma membrane anion transporter in pollen.
Descrição: Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015
URI: http://hdl.handle.net/10451/20237
Designação: Doutoramento em Biologia
Aparece nas colecções:FC - Teses de Doutoramento

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