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Potassium (K) is one of the major nutrients that is essential for plant growth and development. The majority of cellular K+ resides in the vacuole and tonoplast K+ channels of the TPK (Two Pore K) family are main players in cellular K+ homeostasis. All TPK channels were previously reported to be expressed in the tonoplast of the large central lytic vacuole (LV) except for one isoform in Arabidopsis that resides in the plasma membrane. However, plant cells often contain more than one type of vacuole that coexist in the same cell. We recently showed that two TPK isoforms (OsTPKa and OsTPKb) from Oryza sativa localise to different vacuoles with OsTPKa predominantly found in the LV tonoplast and OsTPKb primarily in smaller compartments that resemble small vacuoles (SVs). Our study further revealed that it is the C-terminal domain that determines differential targeting of OsTPKa and OsTPKb. Three C-terminal amino acids were particularly relevant for targeting TPKs to their respective endomembranes. In this addendum we further evaluate how the different localisation of TPKa and TPKb impact on their physiological role and how TPKs provide a potential tool to study the physiology of different types of vacuole.

Potassium (K+) is a major nutrient for plant growth and development. Vacuolar K+ ion channels of the two-pore K+ (TPK) family play an important role in maintaining K+ homeostasis. Several TPK channels were previously shown to be expressed in the lytic vacuole (LV) tonoplast. Plants also contain smaller protein storage vacuoles (PSVs) that contain membrane transporters. However, the mechanisms that define how membrane proteins reach different vacuolar destinations are largely unknown. The Oryza sativa genome encodes two TPK isoforms (TPKa and TPKb) that have very similar sequences and are ubiquitously expressed. The electrophysiological properties of both TPKs were comparable, showing inward rectification and voltage independence. In spite of high levels of similarity in sequence and transport properties, the cellular localization of TPKa and TPKb channels was different, with TPKa localization predominantly at the large LV and TPKb primarily in smaller PSV-type compartments. Trafficking of TPKa was sensitive to brefeldin A, while that of TPKb was not. The use of TPKa:TPKb chimeras showed that C-terminal domains are crucial for the differential targeting of TPKa and TPKb. Site-directed mutagenesis of C-terminal residues that were different between TPKa and TPKb identified three amino acids that are important in determining ultimate vacuolar destination.

The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. To date, the techniques that are available to measure cGMP in plants are limited by low spatial and temporal resolution. In addition, tissue destruction is necessary. To circumvent these drawbacks we have used the δ-FlincG fluorescent protein to create an endogenous cGMP sensor that can report cellular cGMP levels with high resolution in time and space in living plant cells. δ-FlincG in transient and stably expressing cells shows a dissociation constant for cGMP of around 200 nm giving it a dynamic range of around 20-2000 nm. Stimuli that were previously shown to alter cGMP in plant cells (nitric oxide and gibberrellic acid) evoked pronounced fluorescence signals in single cells and in root tissues, providing evidence that δ-FlincG reports changes in cellular cGMP in a physiologically relevant context., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)