Plasma Membrane Proteomics and Functional Characterization & Expression Profiling of Plant Aquaporins

Abstract: In order to identify integral and peripheral proteins associated with the plasma membrane (PM), highly purified Arabidopsis PMs were analyzed by mass spectrometry. In total, 238 putative PM proteins were identified, of which 114 were predicted to have transmembrane domains or to be PM-anchored. Identified proteins were involved in transport, signal transduction, membrane trafficking and stress responses. Almost 25% of the proteins were functionally unclassified and more than half of these were predicted to be integral. In this proteomic study, ten aquaporin (AQP) isoforms were identified. AQPs (also referred to as major intrinsic proteins, MIPs) are integral proteins that facilitate transport of water and other small neutral solutes. In plants, AQPs form a large family with 35 members in Arabidopsis. The number of genes is about the same in moncots. Based on sequence similarity, plant AQPs are divided in four subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic proteins (NIPs) and small basic intrinsic proteins (SIPs). By in-house designed cDNA microarrays and quantitative PCR, the expression patterns of all 35 Arabidopsis AQP genes were monitored in leaves, roots and flowers. This showed that many AQP genes were pre-dominantly expressed in either root or flower organs, but none seemed to be leaf-specific. Most PIP and some TIP genes had a high level of expression, while NIP genes were present at a low level. During gradual drought stress of soil-grown plants, PIP transcripts were generally down-regulated in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which were up-regulated. AtPIP2;6 was constitutively expressed. These three PIP genes were therefore further characterized by promoter-beta-glucuronidase (GUS)- and green florescence protein (GFP)-fusions. Also, expression profiling upon drought stress of PIP genes in different Arabidopsis ecotypes was done. No major differences in expression between ecotypes were detected. Instead expression of many PIP genes correlated with plant water content. Since little is known about the role of AQPs in the elongation zone of growing leaves, an AQP, HvPIP1;6, was cloned from this tissue and characterized by expression in Xenopus oocytes, in-situ PCR and Northern and Western blotting. The gating of the AQP SoPIP2;1 is caused by phosphorylation at Ser115 and Ser274. Two protein kinases acting on these two sites were purified and characterized. The Ser115 kinase was soluble and Ca2+- independent, whereas the Ser274 kinase was PM-associated and Ca2+-dependent.