In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots

We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures.We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs.Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins, small integral membrane proteins belonging to the major intrinsic protein (MIPs) family. Aquaporins form channels that facilitate the movement of water, small uncharged solutes (glycerol, urea, boric acid, silicic acid, hydrogen peroxide) and gases (ammonia, carbon dioxide) across biological membranes. (For recent reviews see [1,2]). TIPs have been either detected, or predicted to localise, to the tonoplast [3].The Arabidopsis genome encodes 10 TIP isoforms [4], further classified into five subgroups: three γ-TIP (TIP1), three δ-TIP (TIP2), the seed-specific α- and β-TIP (TIP3;1 and TIP3;2), one ε-TIP (TIP4;1) and one ζ-TIP (TIP5;1).Several TIP isoforms have been studied in detail as regards their expression [3,5,6] and function [7,8]. TIPs have also been widely employed as intracellular markers for vacuolar biogenesis and identity. Immunofluorescence experiments in root tips and mature embryos of different plant species led to the identification of separate vacuolar compartments within the same cell [9-13]. These experiments indicated an association of γ-TIP