The plant plasma membrane barrier can express aquaporins (PIP1 and PIP2) that show two intriguing aspects: (1) the potential of modulating whole membrane water permeability by co-expression of both types, which have recently been distinguished for showing a different capacity to reach the plasma membrane; and (2) the faculty to reduce water permeation through the pore after cytosolic acidification, as a consequence of a gating process. Our working hypothesis is that these two key features might enhance plasticity of the membrane water transport capacity if they jointly trigger any cooperative interaction. In previous work, we proved by biophysical approaches that the plasma membrane of the halophyte Beta vulgaris storage root presents highly permeable aquaporins that can be shut down by acidic pH. Root Beta vulgaris PIPs were therefore subcloned and expressed in Xenopus oocytes. Co-expression of BvPIP1;1 and BvPIP2;2 not only enhances oocyte plasma membrane water permeability synergistically but also reinforces pH inhibitory response from partial to complete shut down after cytosolic pH acidification. This pH dependent behavior shows that PIP1-PIP2 co-expression accounts for a different pH sensitivity in comparison with PIP2 expression. These results prove for the first time that PIP co-expression modulates the membrane water permeability through a pH regulatory response, enhancing in this way membrane versatility to adjust its water transfer capacity.

Many plasma membrane channels form oligomeric assemblies, and heterooligomerization has been described as a distinctive feature of some protein families. In the particular case of plant plasma membrane aquaporins (PIPs), PIP1 and PIP2 monomers interact to form heterotetramers. However, the biological properties of the different heterotetrameric configurations formed by PIP1 and PIP2 subunits have not been addressed yet. Upon coexpression of tandem PIP2-PIP1 dimers in Xenopus oocytes, we can address, for the first time to our knowledge, the functional properties of single heterotetrameric species having 2:2 stoichiometry. We have also coexpressed PIP2-PIP1 dimers with PIP1 and PIP2 monomers to experimentally investigate the localization and biological activity of each tetrameric assembly. Our results show that PIP2-PIP1 heterotetramers can assemble with 3:1, 1:3, or 2:2 stoichiometry, depending on PIP1 and PIP2 relative expression in the cell. All PIP2-PIP1 heterotetrameric species localize at the plasma membrane and present the same water transport capacity. Furthermore, the contribution of any heterotetrameric assembly to the total water transport through the plasma membrane doubles the contribution of PIP2 homotetramers. Our results also indicate that plasma membrane water transport can be modulated by the coexistence of different tetrameric species and by intracellular pH. Moreover, all the tetrameric species present similar cooperativity behavior for proton sensing. These findings throw light on the functional properties of PIP tetramers, showing that they have flexible stoichiometry dependent on the quantity of PIP1 and PIP2 molecules available. This represents, to our knowledge, a novel regulatory mechanism to adjust water transport across the plasma membrane.

Aquaporins (AQPs) are a family of channel proteins, which transport water and/or small solutes across cell membranes. AQPs are present in Bacteria, Eukarya, and Archaea. The classical AQP evolution paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Soto et al. Gene 503:165-176, 2012). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the AQP community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of AQP data integration in the understanding of water and solute transport in eukaryotic cells.