Electrophysiological Analysis of Metal Transporters and Membrane Transport in Metal Hyperaccumulator Plants
Final Report Abstract
In this grant an electrophysiological approach was used to investigate the role of vacuolar transport processes in metal tolerance and hyperaccumulation in plants. The main part focussed on the functional properties of vacuolar metal transport proteins of the Cation Diffusion Facilitator family. Voltage-clamp studies on Xenopus oocytes after heterologous expression revealed zinc-induced ionic currents. Based on our results obtained from pH-sensitive microelectrodes, ion exchange and inhibitor studies we concluded that the observed zinc-dependent currents did not reflect MTP1-mediated zinc transport, but rather the inhibition of zinc-sensitive background currents. In a second approach we applied the patch-clamp technique to vacuoles isolated from MTP-overexpressing plants. However, in neither of the conditions tested a difference could be detected compared to control plants. Finally, a combined patch-clamp and fura-2 fluorescence approach to monitor exclusively the flux of zinc into the vacuole, initiated by the study of calcium transport through the Slow Vacuolar channel, was applied to vacuoles from MTP-overexpressing plants, but did not give conclusive results within the period of the grant. As part of the second part of the project, channel-mediated ionic currents across the vacuolar membrane of metal hyperaccumulator species were measured using the patch-clamp technique. The response of Slow Vacuolar channels to metal ions was tested both from the cytosolic and lumenal membrane side. Malate currents were studied in terms of current density and sensitivity towards lumenal metal ions. Our expertise on vacuolar malate currents has also been used to contribute to the characterisation of two putative malate channel proteins from Arabidopsis thaliana, in collaboration with an external partner. Although the primary goal to characterise the functional properties of MTP-like metal transport proteins could not be reached, the results of our complementary studies entered in four publications.
Publications
- (2007) The Arabidopsis vacuolar malate channel is a member of the ALMT family. Plant Journal 52, 1169-80
Kovermann, P., Meyer, S., Hortensteiner, S., Picco, C., Scholz-Starke, J., Ravera, S., Lee, Y., Martinoia, E.
- (2008) Fluorescence combined with excised patch: measuring calcium currents in plant ion channels. Plant Journal
Gradogna, A., Scholz-Starke, J., Gutla, P.V.K., Carpaneto, A.
(See online at https://doi.org/10.1111/j.1365-313X.2008.03762.x) - (2009) Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant Alyssum bertolonii. European Biophysics Journal
Corem, S., Carpaneto, A., Soliani, P., Cornara, L., Gambale, F., Scholz-Starke, J.
(See online at https://doi.org/10.1007/s00249-008-0400-2)
