Regulation of aquaporins

One mechanism of post-translational regulation of aquaporin activity is reversible phosphorylation (Fig. 4). Phosphorylation of plant MIPs can increase water permeability (Maurel et al. 1995, Johansson et al. 1998, Guenther et al. 2003). In spinach leaves, SoPIP2;1 is de-phosphorylated under drought stress and therefore inactivated (Johansson et al. 1996). De-phosphorylation occurs at two highly conserved serine residues, Ser115 in cytosolic loop B and, Ser274, in the C terminus, by a Ca2+ dependent protein kinase (Johansson et al 1996, Jo-hannson et al 1998). A number of residues in loop D of SoPIP2;1 have been identified as being involved in gating of the channel (Tornroth-Horsefield et al. 2006). Structural studies show that in the closed conformation, loop D, which has an additional 4-7 amino acid residues in the PIP subfamily, caps the pore of the aquaporin occluding the pore from the cytosol (Tornroth-Horsefield et al. 2006, Fig. 4).

Plant Aquaporins

Fig. 4. The structure of a typical plant aquaporin, with six transmembrane helices (1-6) and five connecting loops (A-E). Shown are the highly conserved NPA motifs; histidine (H) residue, involved in cytosolic pH sensing; two phosphorylation sites [serine (S) residues] in the loop B and C-terminal tail of aquaporins of the PIP2 subgroup (from Luu and Maurel, 2005 and Tornroth-Horsefield et al., 2006).

Fig. 4. The structure of a typical plant aquaporin, with six transmembrane helices (1-6) and five connecting loops (A-E). Shown are the highly conserved NPA motifs; histidine (H) residue, involved in cytosolic pH sensing; two phosphorylation sites [serine (S) residues] in the loop B and C-terminal tail of aquaporins of the PIP2 subgroup (from Luu and Maurel, 2005 and Tornroth-Horsefield et al., 2006).

Plasma-membrane water permeability is regulated by cytosolic pCa (free calcium ion concentration) and pH. Measurements on plasma-membrane vesicles from Beta vulgaris storage roots has revealed very high water permeabilities (>500 ^m s-1), strongly regulated by pCa and pH (Alleva et al. 2006). Arabidopsis Lpcell was reduced by 35% and 69% in the presence of magnesium and calcium ions, respectively (Gerbeau et al. 2002). Both PIP1 and PIP2 aquaporins have a histidine residue (His 197) that appears to be the pH sensitive residue (Tournaire-Roux et al. 2003) (Fig. 4). This may explain the sudden reduction in root Lp when roots are subject to anoxic stress due to a decrease in cytoplasmic pH (Tournaire-Roux et al. 2003).

There is also evidence for mechano-sensitive gating of aquaporins. Large pressure pulses (greater than 0.1 MPa) decreased the Lpcell of maize root cortical cells (Wan et al. 2004). Aquaporin activity, based on Lpcell in Chara, was also reduced in the presence of high concentrations of osmotic solutes and more strongly with increasing size of these solutes (Ye et al. 2005). The water permeability of the symbiosome membrane containing NOD26 also seems to be gated by osmotic solutes (Vandeleur et al. 2005). Hydroxyl radicals may gate aquaporins directly or indirectly but the mechanism involved is unclear (Henzler et al. 2004, Ye and Steudle 2006).

Regulation of aquaporin activity may also occur by interactions between different aquaporins, either in the membrane or via targeting to the membrane (Fetter et al. 2004). The co-expression of ZmPIP1;2, which has low activity, with ZmPIP2;1, ZmPIP2;4 or ZmPIP2;5 increased the osmotic water permeability ofXenopus oocytes. This has also been observed for two grapevine aquaporins, VvPIP1;1 and VvPIP2;2 (Vandeleur et al. 2008). In living maize cells it appears that a physical interaction with a PIP2 is required to traffic PIP1 from the endoplasmic reticulum to the plasma membrane (Zelazny et al. 2007). Redistribution of aquaporins via endomembrane vesicles is also a means of regulating water/solute permeability and this has been shown to occur in response to osmotic stress (Vera-Estrella et al. 2004).

Transcriptional regulation of aquaporins is also linked to control of water transport via changes in channel density in the target membranes. Expression of aquaporins varies diurnally and in response to environmental or developmental influences. Localizations of expression of particular aquaporins can be general or specific for certain cell types and organs. The expression of a PIP1 aquaporin was greater in the endodermis of Arabidopsis root than in its cortex (Schaffner 1998). High expression levels of PIPs generally occur in regions of concentrated water flow (Hachez et al. 2006, Vandeleur et al. 2008). The immuno-localiza-tion examples in Fig. 5 used an antibody raised to a peptide sequence that will detect all PIPls in Vitis. Cells in the exodermis, cells closely associated with the xylem vessels, and cells in phloem bundles showed strong antibody signals (red fluorescence) when compared to pre-immune serum controls.

Diurnal fluctuations in the expression of MIPs have been observed (Ya-mada et al. 1997, Henzler et al. 1999, Lopez et al. 2003, Sakurai et al. 2005) and in some cases these have been correlated with water transport (Henzler et al. 1999, Moshelion et al. 2002, Vandeleur et al. 2008). Changes in expression in leaves during the day have been correlated with leaf water potential (Yamada et al. 1997). The expression of ZmTIP2-3 in maize began to increase just prior to the light period and was at its greatest after four hours of light (Lopez et al. 2003). Diurnal variation continued for some time in continuous darkness. Oryza sativa PIP2 genes, OsPIP1;2 and OsPIP1;3, showed diurnal fluctuations in roots, peaking three hours after the onset of light and decreasing to a minimum three hours after the onset of darkness (Sakurai et al. 2005).

Aquaporins have been shown to be up- or down-regulated in response to many environmental factors such as water stress, salinity, anoxia, nutrient depletion, hormones, low temperature and light (reviewed in Bramley et al. 2007a).

Grenache PI P1 control bright field

Grenache PI P1 control bright field

Chardorinay PIP1 control bright field
Plant Aquaporins
Fig. 5. Transverse sections of a Grenache root (top, showing stele) and Chardonnay root (bottom showing outer cortex and exodermis) showing immunolocalization of VvPIPl proteins. Control was with pre-immune serum. Scale bar = 100 ^m.

5.3. Grapevine aquaporins

In the published literature, ten putative aquaporins with homology to Arabidopsis MIPs have been identified in grapevine (Baiges et al. 2001, Picaud et al. 2003). To date, there are 83 MIPs annotated in Genbank, 73 from the wine grape Vitis vinifera, eight from the rootstock Richter-110 (Vitis berlandieri x Vitis rupestris) and one from the Chinese Wild Grape (Vitis pseudoreticulata). Aquaporins identified from the grapevine Vitis vinifera are from a number of cultivars including Syrah, Cabernet Sauvignon, Pinot noir and Nebbiolo.

The first study of grapevine aquaporins, reported the identification of 8 aquaporins, 5 homologous to the PIP subgroup and 3 to the TIP subgroup (Baiges et al. 2001). The cDNAs encoding aquaporins from Vitis rootstock Richter-110 were obtained by screening a leaf cDNA library with homologous probes and by using reverse Northerns. Tissue specific differential expression patterns were analysed for each of the putative aquaporins (Baiges et al. 2001). Several aquaporins have been identified from grape berries and expression patterns during development vary for the individual isoforms (Picaud et al. 2003, Fouquet et al. 2008).

We have constructed a Vitis vinifera cv Cabernet Sauvignon cDNA library from root and shoot tissues (Shelden 2007). The library was screened for members of the PIP and TIP subfamilies with homologous probes. The library screen resulted in the identification of 13 aquaporin cDNAs 11 of which are full length sequences. Of the cDNAs identified, five are PIP2 aquaporins, six PIP1 aquaporins and two TIP aquaporins. Recently, two high quality draft genome sequences of grapevine; a homozygous line PN40024, bred from Vitis vinifera cv Pinot noir (Jaillon et al. 2007) and a heterozygous grapevine cultivar Vitis vinifera cv Pinot noir clone ENTAV 115 (Velasco et al. 2007) have been reported. Our in silico analysis of the recently sequenced Vitis vinifera clone PN40024 suggests at least 24 full-length MIP genes may be present in the grapevine genome, comprising four subfamilies (Shelden et al, unpublished).

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