Tobacco two-pore calcium channel 1a is localised at the tonoplast, but acts on events at the plasma membrane

Calcium is a central signal regulating a plethora of cellular events. Specificity is brought about by spatio-temporal patterns, so-called signatures that are established by the activity of calcium channels residing in the membranes of different compartments. The role of two-pore calcium channels (TPC) for such signatures has been debated controversially, because evidence for localisation in both, the plasma membrane as well as in the tonoplast, has been proposed. Using a GFP fusion of the tobacco homologue NtTPC1A in the background of tobacco BY-2 cells, we show that this channel is localised at the tonoplast. This localisation depends on actin filaments, but not on microtubules, as shown by pharmacological interference. Since the construct is driven by the constitutive Cauliflower Mosaic Virus 35S promoter, we can also detect phenotypic differences, such as impaired auxin-dependent cell elongation, reduced intracellular calcium content (that can be rescued by supplementation of calcium), and partial resistance to gadolinium, inhibitors of calcium influx. We also monitored the response to harpin, an elicitor from the phytopathogenic bacterium Erwinia amylovora. Here, the overexpressor line shows a higher sensitivity indicating that NtTPC1Aparticipates in defence-related programmed cell death. The data are discussed with respect to a role of NtTPC1A for spatial calcium signatures, and the regulation of cell growth by actin and auxin.