The group IV-A cyclic nucleotide-gated channels, CNGC19 and CNGC20, localize to the vacuole membrane in Arabidopsis thaliana

Abstract

Plant cyclic nucleotide-gated channels (CNGCs) are implicated in the uptake of both essential and toxic cations, Ca 2+ signalling, and responses to biotic and abiotic stress. The 20 CNGC paralogues of Arabidopsis are divided into five evolutionary groups. Group IV-A is highly isolated and consists only of two closely spaced genes, CNGC19 and CNGC20. Prior studies have shown that both genes are induced by salinity and biotic stress. A unique feature of CNGC19 and CNGC20 is their long hydrophilic N-termini. To determine the subcellular locations of CNGC19 and CNGC20, partial and full-length fusions to GFP(S65T) were generated. Translational fusions of the N- termini of CNGC19 (residues 1-171) and CNGC20 (residues 1-200) to GFP(S65T) were targeted to punctate struc- tures when transiently expressed in leaf protoplasts. In the case of CNGC20, but not CNGC19, the punctate structures were co-labelled with a marker for the Golgi. The full-length CNGC19-GFP fusion co-localized with markers for the vacuole membrane (aTIP- and gTIP-mCherry). Vacuole membrane labelling by the full-length CNGC20-GFP fusion was also observed, but the signal was weak and accompanied by numerous punctate signals that did not co-localize with aTIP- or gTIP-mCherry. These punctate structures diminished, and localization of full-length CNGC20-GFP to the vacuole increased, when it was co-expressed with the full-length CNGC19-mCherry. Vacuole membrane labelling was also detected in planta via immunoelectron microscopy using a CNGC20-antiserum on cryopreserved ultrathin sections of roots. We hypothesize that the role of group IV-A CNGCs is to mediate the movement of cations between the central vacuole and the cytosol in response to certain types of abiotic and biotic stress.