Permeability profiling of all 13 Arabidopsis PIP aquaporins using a high throughput yeast approach

Abstract

Plant aquaporins have many more functions than just transporting water. Within the diversity of plant aquaporins are isoforms capable of transporting signaling molecules, nutrients, metalloids and gases. It is established that aquaporin substrate discrimination depends on combinations of factors such as solute size, pore size and polarity, and post-translational protein modifications. But our understanding of the relationships between variation in aquaporin structures and the implications for permeability is limited. High-throughput yeast-based assays were developed to assess diverse substrate permeabilities to water, H₂O₂, boric acid, urea and Na⁺. All 13 plasma membrane intrinsic proteins (PIPs) from Arabidopsis (AtPIPs) were permeable to both water and H₂O₂, although their effectiveness varied, and none were permeable to urea. AtPIP2 isoforms were more permeable to water than AtPIP1s, while AtPIP1s were more efficient at transporting H₂O₂ with AtPIP1;3 and AtPIP1;4 being the most permeable. Among the AtPIP2s, AtPIP2;2 and AtPIP2;7 were also permeable to boric acid and Na⁺. Linking AtPIP substrate profiles with phylogenetics and gene expression data enabled us to align substrate preferences with known biological roles of AtPIPs and importantly guide towards unidentified roles hidden by functional redundancy at key developmental stages and within tissue types. This analysis positions us to more strategically test in planta physiological roles of AtPIPs in order to unravel their complex contributions to the transport of important substrates, and secondly, to resolve links between aquaporin protein structure, substrate discrimination, and transport efficiency.