A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots

Abstract

Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization-time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation.

The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics

“…Western-blot analysis with specific PIP1 and PIP2 antibodies was used to detect the abundance of PIP1 and PIP2 proteins in the 35S:mir1 plants (Santoni et al, 2003). Reduced levels of PIP1 proteins were observed in both 35S:mir1 lines, although (and in accordance with the observed RNA expression levels) a greater decrease was observed in the 35S:mir1-8 line (Supplemental Fig.…”

“…The blot was blocked for 1 h in a modified phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 1% (w/v) bovine serum albumin (PBSTB) and then incubated at room temperature (2 h) or at 4°C (overnight) in the presence of the primary antibodies, anti-PIP1 (AtPIP1;1-AtPIP1;4) and anti-PIP2 (AtPIP2;1-AtPIP2;3), diluted 1:5,000 (Santoni et al, 2003). After washing (2 3 10 min) in PBSTB, the blot was incubated for 1 h with a peroxidaselabeled secondary antibody at 1:20,000 dilution in PBSTB.…”

Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells

“…To investigate the protein content of PM DRMs, we used antibodies raised against three putative protein markers of DRMs (Mongrand et al, 2004;Borner et al, 2005): the H 1 -ATPase PMA2 (Maudoux et al, 2000;Lefebvre et al, 2004), the aquaporin PIP1 (Santoni et al, 2003;Boursiac et al, 2005), and the remorin (Bariola et al, 2004). Figure 1A shows the enrichment of these markers in PM DRMs compared to the PM.…”

“…The antibodies raised against the ATPase PMA2 (Maudoux et al, 2000) may recognize PMA4 as well (Lefebvre et al, 2004). The anti-PIP1 antibody may recognize both the PIP1 and PIP2 families (Santoni et al, 2003). Equal amounts of proteins were loaded in each lane.…”

“…The anti-PMA2 antibody may recognize PMA4 as well (Lefebvre et al, 2004). The anti-PIP1 antibody was raised against a 42-amino acid N-terminal peptide of PIP1-1, and due to sequence homologies, may recognize the PIP1 and PIP2 families (Santoni et al, 2003). The immunolabeling with the different antibodies was carried out at room temperature; the polyvinylidene difluoride membranes were sequentially incubated, at room temperature, with the saturation solution (phosphate-buffered saline, 1% BSA) for 1 h, with IgGs (1/8,000 or 1/4,000) for 2 h, and with anti-IgG-peroxidase conjugate (1/10,000) for 30 min.…”