ANRV342-PP59-24 ARI 2 April 2008 9:6 For instance, a homolog of the bacterial glyc- erol facilitator GlpF has been acquired by the moss Physcomitrella patens by horizontal gene transfer (45), and the genome of this organ- ism and some higher plants (such as poplar) encodes a fifth class of aquaporins, which are closely related to but yet clearly distinct from PIPs (139 U. Johanson, personal communi- cation). Subcellular localization. Plant aquaporins localize in all subcellular compartments form- ing or derived from the secretory pathway. This broad localization pattern reflects the high degree of compartmentation of the plant cell and the need for the cell to control wa- ter and solute transport not only across the plasma membrane but also across intracellu- lar membranes. Similar to PIPs, some NIPs localize in the plasma membrane (82, 134). By contrast, the three Arabidopsis SIP homologs reside mainly in the endoplasmic reticulum (56). However, aquaporins cannot simply be assigned to homogeneous subcellular com- partments. For instance, immunocytochemi- calstudiesusingisoform-specificanti-TIPan- tibodies revealed that distinct types of vacuole that can coexist in the same cell are equipped with specific combinations of TIP isoforms TIP1 and TIP2 isoforms are preferentially associated with the large lytic vacuoles and vacuoles accumulating vegetative storage pro- teins, respectively (59). More recently, Ara- bidopsis thaliana AtTIP1 1 was shown to accu- mulate in spherical structures named bulbs, tentatively identified as intravacuolar invagi- nations made of a double tonoplast membrane (118). Preferential expression of PIPs in plas- malemmasomes (convoluted plasma mem- brane invaginations that dip into the vacuole) has also been observed in Arabidopsis leaves (116). Finally, preferential expression of a PIP and a NIP homolog on the distal side of root exo- and endodermal cells has been described in maize and rice, respectively (46, 82). Such cell polarization is consistent with the uptake and centripetal transport of water and solute Plasmalemmasome: convoluted plasma membrane invagination in roots (see below). A future challenge is to understand how aquaporins can be specif- ically targeted to membrane subdomains in the plant cell and how targeting contributes to their functional specialization. Mechanisms of Transport Pore structure and transport mecha- nisms. X-ray crystallography determination of atomic structures of microbial, animal, and plant homologs points to highly con- served structural features in the aquaporin family (38, 137). Aquaporins are 23���31 kDa proteins comprising six membrane-spanning domains tilted along the plane of the mem- brane and linked by five loops (A to E ) lo- cated on the intra- (B, D) or extracytoplas- mic (A, C, E ) side of the membrane. The N- and C-terminal extremities are both ex- posed to the cytosol (Figure 1). A central aqueous pore is delineated by the transmem- brane domains and loops B and E, which both carry a conserved Asn-Pro-Ala (NPA) motif and dip from either side of the membrane into the center of the molecule. Projection structures determined by cryo-electron mi- croscopy indicate that, similar to their animal and microbial counterparts, PIPs and TIPs occur as tetramers in their native membranes (24, 34). X-ray structures have confirmed this type of assembly (38, 137) and in combi- nation with molecular dynamics simulations have provided critical insights into the funda- mental principles of aquaporin transport se- lectivity (38, 133) (Figure 1). In brief, the substrate specificity of aquaporins can be ex- plained by several mechanisms, including size exclusion at two main pore constrictions [aro- matic/Arg (Ar/R) and NPA] and stereospe- cific recognition of the substrate mediated by spatially defined H-bonding and hydropho- bic interactions within the pore. The remark- able impermeability of aquaporins to protons is explained by electrostatic repulsion, dipole orientation, and transient isolation of the water molecule as it passes within a single www.annualreviews.org ��� Plant Aquaporins 597 Annu. Rev. Plant Biol. 2008.59:595-624. Downloaded from arjournals.annualreviews.org by Volcani Institute of Agriculture Research on 12/11/08. For personal use only.

ANRV342-PP59-24 ARI 2 April 2008 9:6 a Phe81 Asn101 Arg225 His210 Asn222 b Figure 1 Representative atomic structure of a plant aquaporin (a) and general molecular mechanisms of transport selectivity (b). (a) Structure of the open conformation of Spinacia oleracea plasma membrane intrinsic protein 2 1 (SoPIP2 1) [Protein Data Bank (PDB) ID 2B5F] (137) showing a typical tetrameric arrangement. Each monomer is composed of six tilted transmembrane helices the N-terminal (red ) and C-terminal ( green) helices of the top left monomer are shown. The pores of individual monomers are emphasized by the space-filling representation of the three other monomers. (b) The two highly conserved Asn-Pro-Ala (NPA) motifs (represented by Asn101 and Asn222, green) are in close proximity to form one of the main pore constrictions. Another constriction called Ar/R (red ) is formed on the extracytoplasmic side of the membrane by a spatial arrangement of aromatic (Ar) residues, such as Phe81 and His210, facing an Arg (R) residue, here Arg225. Proton transport is blocked by electrostatic repulsion in the Ar/R constriction and the dipole orientation of the water molecule by the two Asn residues of the NPA motifs. This results in a transient isolation of the water molecule within the single file of water molecules that fills the pore (orange spheres). file of water molecules through the center of the pore (11, 38, 133) (Figure 1). The molecular basis of plant aqua- porin selectivity has been investigated more specifically by homology modeling of pore structures at the Ar/R constriction (8, 150). Analysis of all 35 Arabidopsis homologs yielded up to nine pore types (150) and additional types exist in maize and rice (8). Whereas all PIPs exhibit a narrow pore structure typ- ical of orthodox, water-selective aquaporins, larger substrate specificity was predicted for other plant homologs. According to this anal- ysis, AtNIP6 1 belongs to one of two NIP subgroups and as such exhibits a low and high permeability to water and urea, respec- tively (151). An Ala119Trp substitution, made to mimic the pore configuration of mem- bers of the other NIP subgroup, also con- fers novel permeability properties, i.e., higher permeability to water and failure to trans- port urea. This result and other examples in animal aquaporins (11) show that point mu- tations can drastically alter transport speci- ficity and that these proteins may be engi- neered to accommodate novel substrates of interest. Transport assays and aquaporin sub- strates. Functional expression in Xenopus oocytes or yeast was essential to show that plant MIP homologs of all four subclasses can function as water channels (56, 66, 94, 115). Enhanced water permeability of pro- teoliposomes containing a purified aquaporin provides the ultimate proof of water channel activity. Such functional reconstitution has been performed with GmNOD26 purified 598 Maurel et al. Annu. Rev. Plant Biol. 2008.59:595-624. Downloaded from arjournals.annualreviews.org by Volcani Institute of Agriculture Research on 12/11/08. For personal use only.

ANRV342-PP59-24 ARI 2 April 2008 9:6 from native peribacteroid membranes (27) or after production of Spinacia oleracea SoPIP2 1 in Pichia pastoris (67). Although strict compar- ative measurements have not been performed in plants, plant aquaporins may, similar to their animal homologs, exhibit marked differ- ences (up to 30-fold) in intrinsic water trans- port activity (154). Expression studies in Xenopus oocytes also show that, similar to animal and bacterial aquaglyceroporins, some plant aquaporin iso- forms can transport small neutral solutes such as glycerol (12), urea (42), formamide, ac- etamide (115), methylammonium (53), boric acid (134), silicic acid (82), or lactic acid (20). Ammonia (NH3) and CO2 transport is de- tected using substrate-induced extra- and in- tracellular acidification, respectively, whereas ammonium (NH4+) transport by Triticum aes- tivum TaTIP2 1 results in inward currents (53, 144). Finally, expression in yeast cells de- ficient in endogenous systems responsible for urea or hydrogen peroxide uptake has proved efficient to screen, on the basis of a survival assay, aquaporin isoforms that possibly trans- port these molecules these properties are sub- sequently confirmed by true transport assays (13, 77). Several approaches have established that aquaporins contribute significantly to the per- meability of plant membranes to water and small neutral solutes. In most studies, mercury derivatives, which act through oxidation and binding to Cys residues, were used as com- mon aquaporin blockers. Plant aquaporins do not have Cys residues at conserved positions and various residues may be involved in plant aquaporin inhibition (23). We also note that mercury-resistantPIPshavebeendescribedin Arabidopsis and tobacco (12, 25). In some stud- ies, the permeability profiles of the vacuolar, peribacteroid, and plasma membranes were characterized by stopped-flow spectropho- tometry on purified membrane vesicles, and mercury induced a marked (50%���90%) inhi- bition of water transport in the first two types of membranes (42, 95, 104, 105, 115). In addi- tion, a good parallel was established between the high permeability of the tobacco tonoplast and soybean peribacteroid membrane to urea and formamide, respectively, and the capacity of Nicotiana tabacum NtTIPa and GmNOD26 to transport these solutes (42, 115). In other studies, the respective water permeabilities of the plasma membrane and the tonoplast and their sensitivity to mercury were in- ferred from independent osmotic swelling assays on protoplasts and isolated vacuoles and calculations using a three-compartment model (92, 99, 102). Figure 2 summarizes the contribution of plant aquaporins to wa- ter and solute transport in multiple subcellular compartments. Molecular Mechanisms of Regulation Cotranslational and posttranslational modifications. Because of their high abun- dance in plant membranes, and despite their high hydrophobicity, some aquaporins have proved to be particularly amenable to biochemical analysis, in comparison with other membrane proteins (34, 48, 63). Pro- teomics, and mass spectrometry techniques in particular, have recently been added to more classical techniques to produce a thorough description of aquaporin co- and posttrans- lational modifications (26, 121, 122). For instance, N-terminal maturation of PIP1s and PIP2s occurs through N-��-acetylation or cleavage of the initiating residue, respectively (121). In vivo and in vitro labeling studies, experiments with antiphosphopeptide anti- bodies, and mass spectrometry analyses have provided direct evidence for phosphorylation of Ser residues in the N-terminal and C- terminal tails of Phaseolus vulgaris PvTIP3 1, GmNOD26, and SoPIP2 1 (26, 43, 63, 64, 96). PIPs show a conserved phosphorylation site in loop B and multiple (up to three) and interdependent phosphorylations occur in adjacent sites of their C-terminal tail (63, 64 S. Prak, S. Hem, J. Boudet, N. Sommerer, G. Viennois, M. Rossignol, C. Maurel & V. Santoni, unpublished results). Purification of calcium-dependent protein www.annualreviews.org ��� Plant Aquaporins 599 Annu. Rev. Plant Biol. 2008.59:595-624. Downloaded from arjournals.annualreviews.org by Volcani Institute of Agriculture Research on 12/11/08. For personal use only.

ANRV342-PP59-24 ARI 2 April 2008 9:6 kinases acting on aquaporins has been undertaken by several laboratories (48, 129). Although most plant aquaporins do not ex- hibit glycosylation, this type of modification has been observed in GmNOD26 and in an ice plant TIP (96, 146). In the latter case, glycosy- lation was required for subcellular redistribu- tion (described below). Aquaporins were also Silicic acid Hydrogen peroxide Water Ammonia? Water Boric acid Water CO2 Ammonia Water Glycerol Urea Water Protein storage vacuole Vegetative protein storage vacuole Lactic acid Water PIP1s PIP2s TIP1s TIP2s TIP3s AtNIP2 1 Lsi1/OsNIP2 1 AtNIP5 1 NOD26 SIPs Nucleus and ER Chloroplast Chloroplast Golgi apparatus Lytic/central vacuole Early endosome Plasmalemmasome Vacuolar bulb Plasma membrane Cell wall Peribacteroid membrane Multivesicular body / late endosome / prevacuolar compartment 600 Maurel et al. Annu. Rev. Plant Biol. 2008.59:595-624. Downloaded from arjournals.annualreviews.org by Volcani Institute of Agriculture Research on 12/11/08. For personal use only.

ANRV342-PP59-24 ARI 2 April 2008 9:6 the first plant membrane proteins found to be methylated (121). For instance, AtPIP2 1 can carryoneortwomethylgroupsonitsLys3and Glu6 residues, respectively. These data show that, in addition to a high isoform multiplic- ity, plant aquaporins occur in a large variety of modified forms, which suggests intricate co- and posttranslational regulation mechanisms. Gating. The gating of aquaporins, i.e., the opening and closing of the pore, can be regulated by multiple factors. A role for phosphorylation in gating PvTIP3 1, GmNOD26, and SoPIP2 1 was first deduced from functional expression in oocytes of these aquaporins, either wild-type or with point mutations at their phosphorylation sites (43, 63, 93), and by using pharmacological al- terations of endogenous protein phosphatases and kinases. A role for phosphorylation in GmNOD26 gating has been unambiguously established by stopped-flow measurements in purified peribacteroid membranes, showing that alkaline phosphatase-mediated dephos- phorylation leads to reduced water perme- ability (43). Water transport measurements in plasma membrane vesicles purified from Arabidopsis suspension cells or Beta vulgaris roots also suggest that PIPs can be gated from the cytosolic side by protons and diva- lent cations (4, 41). A half-inhibition of water Gating: opening and closing of a membrane channel pore transport is observed at ���pH 7.5 and for free Ca2+ concentrations in the 100 ��M range (4, 41). Beet plasma membranes exhibit an additional affinity component in the 10 nM range (4). The molecular bases of aquaporin gating have been elucidated from structure-function analyses in Xenopus oocytes and more re- cently from the atomic structures of SoPIP2 1 in its open and closed conformations (137, 138). These studies established that protons are sensed by a His residue that is perfectly conserved in loop D of all PIPs (138). The molecular mechanisms that lead to a confor- mational change of loop D and occlusion of the pore upon protonation of the His residue or binding of divalent cations are detailed in Figure 3. The atomic structure of SoPIP2 1 also indicates how phosphorylation of loop B wouldunlockloop D toallowtheopenconfor- mation. By contrast, phosphorylation of the C-terminal tail would act in trans to prevent loop D of an adjacent monomer from adopt- ing a closed-pore conformation (137). A role for solutes in gating aquaporins has been proposed, based mainly on pressure probe measurements in Chara cells (155). In- hibition of cell water permeability is linked to the presence of the solute on either side of the membrane and is strongly dependent on so- lute molecular size. A tension/cohesion model ��� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� Figure 2 The multiple cellular functions of plant aquaporins. The figure illustrates the variety of transport functions achieved by aquaporins in various subcellular compartments. The different aquaporin subclasses or isoforms are identified below the illustration in distinct colors. Isoforms of the plasma membrane intrinsic protein 1 (PIP1) and PIP2 subfamilies are thought to follow the secretory pathway, which carries cargo from the endoplasmic reticulum (ER) toward the plasma membrane through the Golgi apparatus. PIPs also undergo repeated cycles of endocytosis and recycling through endosomal compartments before being eventually targeted to the lytic vacuole through the multivesicular body. In Arabidopsis leaves, PIP1s label plasmalemmasomes (116). Tonoplast intrinsic protein 1s (TIP1s) are found in the lytic vacuole membrane. AtTIP1 1 localizes in spherical structures named bulbs in epidermal cells of young cotyledons or salt-treated roots (15, 118). TIP2s and TIP3s are preferentially associated with vacuoles that accumulate vegetative storage proteins and seed protein storage vacuoles, respectively. Nodulin-26���like intrinsic membrane proteins (NIPs) show a broad range of subcellular localization patterns. AtNIP2 1 is localized in the endoplasmic reticulum and the plasma membrane (20, 97), the Oryza sativa silicon influx transporter low silicon rice 1 (Lsi1, also namedOsNIP2 1) and the Arabidopsis thaliana boric acid channel AtNIP5 1 are localized in the plasma membrane, whereas Glycine max nodulin-26 (GmNOD26) is exclusively expressed in the peribacteroid membrane. www.annualreviews.org ��� Plant Aquaporins 601 Annu. Rev. Plant Biol. 2008.59:595-624. Downloaded from arjournals.annualreviews.org by Volcani Institute of Agriculture Research on 12/11/08. For personal use only.