Plant Aquaporins: A study of expression, localization, specificity, and regulation
Abstract: Aquaporins, or Major Intrinsic Proteins (MIPs), are integral proteins that facilitate transport of water and other small neutral solutes across biological membranes. They belong to a well conserved and ancient family of proteins, present in all organisms ranging from bacteria to plants and humans. The aquaporin family in plants is large, indicating complex and regulated water transport within the plant in order to adapt to different environmental conditions. All aquaporin isoforms probably work together in an orchestrated manner, where each individual aquaporin isoform displays a specific localization pattern, substrate specificity, and regulatory mechanism. When analyzing the whole genome of the model plant Arabidopsis thaliana, 35 aquaporin-encoded genes were identified. Based on sequence similarities and by phylogenetic analyses they were divided into four subfamilies; Plasma membrane Intrinsic Proteins (PIPs), Tonoplast Intrinsic Proteins (TIPs), NOD26-like Intrinsic Proteins (NIPs), and Small basic Intrinsic Proteins (SIPs). These subfamilies are conserved in many plant species. Based on the four subfamilies a new uniform nomenclature for all plant aquaporins was proposed, which is now widely accepted and used. A gene expression study, using a DNA microarray and quantitative real-time reverse transcriptase PCR, of all the 35 aquaporin genes in Arabidopsis was performed. The relative amounts of each isoform in leaves, roots, and flowers were analyzed, as well as their individual responses to drought stress. Focusing on four of the nine isoforms in the Arabidopsis NIP subfamily, i.e. AtNIP1;2, AtNIP2;1, AtNIP4;2, and AtNIP6;1, the gene expression on tissue and cell level was studied with promoter::GUS constructs, the protein localization was studied on the subcellular level in different organs with immunoblots, and the permeability to water and glycerol was examined by heterologous expression in Xenopus oocytes. The spinach leaf plasma membrane aquaporin SoPIP2;1 was heterologously overexpressed in the yeast Pichia pastoris, purified, and functionally characterized by reconstitution into proteoliposomes. The water channel activity of SoPIP2;1 has previously been shown to be regulated by phosphorylation of Ser115 and Ser274. Two protein kinases, acting on these two phosphorylation sites in SoPIP2;1, were partly purified and characterized.
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