Anionic currents in pollen grain protoplasts from Arabidopsis thaliana and Lilium longiflorum ; Correntes aniónicas em protoplastos de grãos de pólen de Arabidopsis thaliana e Lilium longiflorum

Media type: E-Book

Title: Anionic currents in pollen grain protoplasts from Arabidopsis thaliana and Lilium longiflorum ; Correntes aniónicas em protoplastos de grãos de pólen de Arabidopsis thaliana e Lilium longiflorum

Contributor: Santos, Ana Bárbara Tavares dos [Author]

Published: [Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2011

Language: English

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University thesis: Dissertation, 2011

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Description: Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2011 ; Upon hydration in a simple medium, pollen grains germinate and develop a cytoplasmic extension designated as the pollen tube, the growth rate of which can reach up to 4 μm s-1, making it one of the fastest polarized cellular growths in nature. This apical growth is oscillatory and its control has been systematically shown to be underlined by ion dynamics, namely ion fluxes across the membrane and cytoplasmatic ionic gradients. These phenomena were identified and characterized by means of imaging techniques and by Self Referencing Vibrating Probes. Thus far the ions known to be involved are Chloride (Cl-), Calcium (Ca2+), Proton (H+) and Potassium (K+), which apical fluxes oscillate with the same frequency as that of the apical growth. Zonia et al. (2002) identified and characterized the Cl- fluxes in growing pollen tubes from two species, Lilium longiflorum and Nicotiana tabacum, showing that this ion leaves the pollen tube through an oscillating efflux at the tip and enters through a non-oscillating influx at the shank of the tube, starting at approximately 15 μm from the tip. These authors also associated the efflux of Cl- to the Ins(3,4,5,6)P4 (Inositol 3,4,5,6 tetra-kis-phosphate) signaling pathway. This compound, known for inhibiting receptor mediated Ca2+ dependent Cl- currents, inhibited the pollen tube growth, induced the increase of the cellular volume and compromised the normal efflux of Cl-. Other inhibitors were also tested: DIDS (4,4'-di-iso-tiocianate-stilbeno-2,2'-disulfonic acid), o NPPB (5-nitro-2-(3-fenil-propil-amino)benzoic acid) and niflumic acid completely inhibited the pollen tube growth, the cytoplasmic streaming and caused the increase of the cellular volume. Nevertheless, only DIDS completely abolished the oscillatory effluxes of Cl-. Zonia et al. (2002) showed that the Cl- fluxes are essential to the pollen tube growth due to its role in the maintenance of the cellular volume. This observation let to the hypothesis that the fluxes of water through the plasma membrane follow the fluxes of Cl-. The work developed in this thesis lay with the isolation and characterization of the anionic currents present in the pollen grain protoplasts of two species, Arabidopsis thaliana and Lilium longiflorum. The first species was chosen due to the fact that it is the biological model for plants, endowing it with the necessary molecular tools for the latter identification of the channel or channels responsible for the Cl- fluxes reported in Zonia et al. (2002). The second species was chosen not only because it has been recurrently used as a model in the study of development of pollen tubes, but also because it would allow for a comparison between the same type of currents in two very different species. The necessity of developing specific protocols for the isolation of the anionic currents in this system stemmed from the fact that, although there was already published reports on anionic currents in plant cell protoplasts, namely from leaf meristem, from stomata and from apical root cells, the existing protocols were inadequate, for producing quality protoplasts and for not allowing the patch clamp technique to be applied. It was necessary to adjust the time of incubation in the enzyme solution, and the time and speed of the centrifugation steps in the protocol for the production and isolation of protoplasts. For the isolation of the anionic currents it was necessary to adjust the osmolarity of the bath and pipette solutions, their ionic content. The anionic currents found in the plasma membrane of pollen grain protoplasts from Arabidopsis thaliana and Lilium longiflorum were characterized by means of the Whole Cell (WC) configuration of the patch clamp technique, under symmetrical anionic concentrations, in which Cl- was the permeable anion in greater concentration. Under these experimental conditions, it was possible to identify, in the protoplasts of Arabidopsis thaliana, three different anionic current populations, all with a strong outward rectification. These were ICl1 (current lost during rundown, after the establishment of the WC configuration), ICl2 (current insensitive to the inhibitor NPPB), and ICl3 (current inhibited by 100 μM of NPPB). ICl2 and ICl3 were measured after rundown. Despite the strong outward rectification, the putative channels responsible for the detected anionic currents allow the passage of current in both directions (inwardly and outwardly), since both negative and positive currents suffered rundown and were inhibited by NPPB. The currents presented a time dependent activation for the more positive membrane potentials (Vm), that is, an activation induced by depolarizing Vm. This can be described by three current components, one which is instantaneous - Iinst (sub millisec) and two which are time dependent - I1 e I2, characterized by the time constants 1 (hundreds of millisec) and 2 (tens of millisec), respectively. Using a tail protocol it was possible to observe that these currents are also characterized by a hyperpolarizing time dependent deactivation. The variation of the cord conductance (G) with Vm was also studied, by fitting a Boltzmann equation to the values of G when plotted against Vm. This analysis revealed that ICl1 has a different sensitivity to variation in Vm. By diminishing the Cl- concentration in the bath solution ([Cl-]out) after the rundown, it was possible, not only to confirm the anionic nature of the currents but also to determine that ICl1 and ICl3 are regulated by [Cl-]out. The ionic selectivity of the two populations of currents was studied by replacing part of the [Cl-]out by NO3-. It was possible to conclude that the permeability to NO3- was two times higher than to Cl- (PNO3-/PCl-). The effect of three different intracellular calcium concentrations ([Ca2+]in, in the nM, μM and mM range), was studied showing that all three currents increased in intensity with increasing [Ca2+]in, while their sensitivity to NPPB decreased. The sensitivity of G to variations in Vm was not changed. The populations of anionic currents - ICl1, ICl2 and ICl3 – with very similar characteristics were also found in pollen grain protoplasts from Lilium longiflorum. These differed, not only in the response to [Ca2+]in, but also in the sensitivity to NPPB, which was inferior, since the minimal NPPB concentration needed was 500 μM. Other differences laid with the PNO3-/PCl-, which in these currents was 1.2, and with the fact that these were not regulated by [Cl-]out. From the values of the currents it was possible to calculate the anionic fluxes that these could originate. By varying the Vm and [Ca2+]in is was possible to oscillate the values of the calculated fluxes in a manner compatible with the observed Cl- fluxes reported by Zonia et al. (2002) in growing pollen tubes from Lilium longiflorum. It was not possible to perform the same comparison for Arabidopsis thaliana, since the Cl- fluxes in growing pollen tubes haven't been characterized in this species yet. Nevertheless it is possible to claim with some level of confidence that the anionic currents here described can be responsible for the observed fluxes. The identification of the channel or channels responsible for the observed anionic currents was also attempted. Through the analysis of the pollen grain transcriptomic it was possible to isolate the following gene candidates: CLC-c, CLC-d, MSL10, ALMT12, SLAH2, SLAH3 and CaCC. The first two genes, CLC-c and CLC-d, were eliminated in a second selection phase, not only because they were both localized in intracellular membranes, namely the tonoplast and the trans-Golgi network, but also because all the CLC channels so far characterized revealed themselves as anion:H+ cotransporters. MSL10 was also eliminated because these channels aren't regulated by cytoplasmic Ca2+ or by Vm. ALMT12 was eliminated because, unlike the currents elicited by this channel, the anionic currents described here suffered a decrease in intensity when 5 mM of malate was added to the bath solution, indicating a smaller permeability to this anion. The candidates SLAH2 and SLAH3, homologues of SLAC1, were not selected because they have yet to be electrophysiologically characterized. The best candidate was CaCC, since the currents in the animal homologues have been characterized, revealing themselves very similar to the ones described in this thesis. Unfortunately it was impossible to confirm this identification due to a lack of a true KO mutant for this gene. ; Ao germinar, o grão de pólen forma uma extensão citoplasmática designada por tubo polínico, cujo crescimento é polarizado e apical, podendo atingir os 4 μm s-1. Este crescimento distingue-se por ser oscilatório e por estar intimamente associado a fluxos iónicos através da membrana plasmática e aos gradientes citoplasmáticos que os fluxos orig...

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