# Aquaporins in Plants.

Christophe Maurel, Yann Boursiac, Doan Luu, Veronique Santoni, Zaigham Shahzad, Lionel Verdoucq

► To cite this version:

Christophe Maurel, Yann Boursiac, Doan Luu, Veronique Santoni, Zaigham Shahzad, et al.. Aquaporins in Plants.. Physiological Reviews, 2015, 95 (4), pp.1321-58. 10.1152/physrev.00008.2015. hal-01282576

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AQUAPORINS IN PLANTS

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3 Christophe Maurel, Yann Boursiac, Doan-Trung Luu, Véronique Santoni, Zaigham Shahzad, Lionel

4 Verdoucq

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6 Biochimie et Physiologie Moléculaire des Plantes, Unité Mixte de Recherche 5004,

7 CNRS/INRA/Montpellier SupAgro/Université de Montpellier, F-34060 Montpellier, Cedex 2, France

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9 Corresponding author:

Dr. Christophe Maurel

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Biochimie et Physiologie Moléculaire des Plantes

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SupAgro/INRA/CNRS/UM UMR 5004

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2, Place Viala

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F-34 060 Montpellier Cedex 2

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France

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Phone: + 33 (0)4 99 61 20 11

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Fax : + 33 (0)4 67 52 57 37

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E-mail : maurel@supagro.inra.fr

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20 TABLE OF CONTENTS

<table><tr><td>21</td><td>I) Introduction</td><td>4</td></tr><tr><td>22</td><td>II) Plant aquaporin diversity</td><td>5</td></tr><tr><td>23</td><td>II.A - Phylogeny of plant aquaporins</td><td>5</td></tr><tr><td>24</td><td>II.B - Plant aquaporin evolution</td><td>5</td></tr><tr><td>25</td><td>III) Molecular functions of plant aquaporins</td><td>6</td></tr><tr><td>26</td><td>III.A - Functional expression and selectivity</td><td>6</td></tr><tr><td>27</td><td>III.B - Gating</td><td>8</td></tr><tr><td>28</td><td>III.C - Posttranslational modifications</td><td>10</td></tr><tr><td>29</td><td>III.D - Heteromerization</td><td>10</td></tr><tr><td>30</td><td>III.E - Perspectives</td><td>11</td></tr><tr><td>31</td><td>IV) Cell biology of plant aquaporins</td><td>11</td></tr><tr><td>32</td><td>IV.A - Subcellular localization patterns</td><td>11</td></tr><tr><td>33</td><td>IV.B - Aquaporin trafficking</td><td>13</td></tr><tr><td>34</td><td>IV.C - Perspectives</td><td>14</td></tr><tr><td>35</td><td>V) Cell-autonomous and intracellular functions</td><td>15</td></tr><tr><td>36</td><td>V.A - Osmoregulation</td><td>15</td></tr><tr><td>37</td><td>V.B - ROS detoxification and signaling</td><td>16</td></tr><tr><td>38</td><td>V.C - Vacuolar storage</td><td>16</td></tr><tr><td>39</td><td>V.D - Chloroplast transport</td><td>16</td></tr><tr><td>40</td><td>VI) Whole plant water transport and transpiration</td><td>17</td></tr><tr><td>41</td><td>VI.A - Context</td><td>17</td></tr><tr><td>42</td><td>VI.B - Root water transport</td><td>17</td></tr><tr><td>43</td><td>VI.C - Leaf water transport</td><td>20</td></tr><tr><td>44</td><td>VI.D - Perspectives</td><td>22</td></tr><tr><td>45</td><td>VII) Plant growth and development</td><td>24</td></tr><tr><td>46</td><td>VII.A - Hydraulics of plant growth</td><td>24</td></tr><tr><td>47</td><td>VII.B - CO₂ fixation</td><td>25</td></tr><tr><td>48</td><td>VII.C - Nutrient allocation and toxicity</td><td>26</td></tr><tr><td>49</td><td>VII.D - Plant reproduction</td><td>27</td></tr><tr><td>50</td><td>VIII) Biotic interactions</td><td>28</td></tr><tr><td>51</td><td>VIII.A - Rhizobium-legume symbiosis</td><td>28</td></tr><tr><td>52</td><td>VIII.B - Mycorrizhae</td><td>29</td></tr><tr><td>53</td><td>VIII.C - New directions: investigating beneficial or pathogenic interactions</td><td>29</td></tr><tr><td>54</td><td>IX) Aquaporins and plant genetic improvement</td><td>30</td></tr><tr><td>55</td><td>IX.A - Success stories and failures</td><td>30</td></tr><tr><td>56</td><td>IX.B - Mechanisms</td><td>31</td></tr><tr><td>57</td><td>IX.C - Targeting micronutrients</td><td>31</td></tr><tr><td>58</td><td>X) Conclusions and perspectives</td><td>32</td></tr><tr><td>59</td><td>X.A - Conclusions</td><td>32</td></tr><tr><td>60</td><td>X.B - Emerging research directions</td><td>32</td></tr><tr><td>61</td><td>XI) References</td><td>33</td></tr><tr><td>62</td><td></td><td></td></tr></table>
63 ABSTRACT

64 Aquaporins are membrane channels that facilitate the transport of water and small neutral
65 molecules across biological membranes of most living organisms. In plants, aquaporins occur as
66 multiple isoforms reflecting a high diversity of cellular localizations, transport selectivity and
67 regulation properties. Plant aquaporins are localized in the plasma membrane, endoplasmic
68 reticulum, vacuoles, plastids and, in some species, in membrane compartments interacting with
69 symbiotic organisms. Plant aquaporins can transport various physiological substrates in addition to
70 water. Of particular relevance for plants is the transport of dissolved gases such as carbon dioxide
71 and ammonia or metalloids such as boron and silicon. Structure-function studies are developed to
72 address the molecular and cellular mechanisms of plant aquaporin gating and subcellular trafficking.

73 Phosphorylation plays a central role in these two processes. These mechanisms allow aquaporin
74 regulation in response to signaling intermediates such as cytosolic pH and calcium, and reactive
75 oxygen species. Combined genetic and physiological approaches are now integrating this knowledge,
76 showing that aquaporins play key roles in hydraulic regulation in roots and leaves, during drought but
77 also in response to stimuli as diverse as flooding, nutrient availability, temperature or light. A general
78 hydraulic control of plant tissue expansion by aquaporins is emerging and their role in key
79 developmental processes (seed germination, emergence of lateral roots) has been established.

80 Plants with genetically altered aquaporin functions are now tested for their ability to improve plant
tolerance to stresses. In conclusion, research on aquaporins delineates ever expanding fields in plant
integrative biology thereby establishing their crucial role in plants.

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# I) INTRODUCTION

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In their large majority, plants are autotrophic organisms that just require light and a carbon source (usually carbon dioxide, CO₂), on the one hand, and water and mineral nutrients, on the other hand, to achieve their life cycle. Because they are sessile, plants have, however, to efficiently absorb water and mineral nutrients from their close surroundings. This function is mostly fulfilled by the root system, which shows a remarkable ability to grow continuously and explore the soil for available resources. With no short-term escape strategies, plants also have to continuously face severe constraints from both the soil and aerial environments. These include abiotic stresses such as a lack or excess of water (drought, flooding), or beneficial or toxic mineral ions at varying concentrations in the soil. High or low extremes in temperature or light intensity also impose severe stresses, on the shoot especially. In addition, plants are under constant attack by a myriad of herbivores and pathogens including viruses, bacteria, insects or fungi. Many plant species are also able to develop symbioses with specific soil micro-organisms which, in particular, optimize plant mineral nutrition. Plants therefore continuously adjust their metabolic functions, growth and development, to adapt to an ever changing abiotic and biotic environment.

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With respect to their water status, plants exhibit remarkable features. Their aerial parts mediate a tricky trade-off with the atmosphere, by absorbing CO₂, a fundamental brick for photosynthesis, and releasing water by transpiration. This exchange is realized and tightly controlled by stomata, microscopic pores located in the epidermis of the plant's aerial parts (FIGURE 1). Transpiration is made possible by an intense flow of water (sap) travelling throughout the plant body, from the roots to the substomatal chambers where it evaporates. This stream is particularly useful to drive water and nutrient ascent to the uppermost parts of the shoots. Whereas long-distance water transport mostly occurs through the xylem, which is formed of specialized dead vessels, water uptake by roots and delivery to shoots requires transport through living tissues (FIGURE 1). Controlling the intensity and direction of these flows is particularly important for maintaining the whole plant water status. At the cellular level, the presence of a cell wall allows buildup of intracellular hydrostatic pressure (turgor) of several atmospheres which largely supports the erect shape of the plant. In addition, plant growth and development are determined by cell divisions in well-defined territories called meristems, and subsequent expansion growth of the living cells. Cell turgor provides the motive force for the latter process. The metabolic activity of the plant and its growth and development are therefore highly dependent on its water status.

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Despite a few pioneering works [reviewed in (185)], the identification of water channel proteins in plants in the early 1990s (187), shortly after their discovery in animals, was fairly unexpected and provided a strong momentum for studies on plant water transport. Early studies of these so-called aquaporins contributed to a general boost in the molecular characterization of membrane transport systems in plants and brought new paradigms to address the molecular bases of plant water relations. After 20 years, plant aquaporins are recognized as multifunctional proteins transporting water but also gases such as CO₂, nutriments [e.g. boron (B), silicon (Si)] or reactive oxygen species (ROS). Thus, aquaporin studies have spread to many fields of plant biology, from molecular membrane biophysics to cell biology and signaling. Their function also appears central for the physiology of plant growth and responses to abiotic stresses. The present review chapter covers the many facets of aquaporin functions in plants, and stresses their importance and functional originality in these organisms.

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## II) PLANT AQUAPORIN DIVERSITY

### II.A - Phylogeny of plant aquaporins

130 Aquaporins are now assimilated in a broad sense to the ancient superfamily of Major Intrinsic Proteins (MIPs). MIPs are present throughout the living kingdom, with an exception for thermophilic Archaea and intracellular bacteria (1). MIP homologues have a reduced (~25%) overall sequence conservation but show a typical sequence signature, with six putative membrane spanning domains and two highly conserved Asn-Pro-Ala (NPA) motifs. In addition, MIP coding sequences are formed from a direct sequence repeat, which creates an internal symmetry of the channel protein, with its two halves (each with 3 transmembrane domains) showing an inverted insertion in the membrane. With increasing number of plant genome sequences available, aquaporin genes have now been fully described in several herbaceous (Arabidopsis thaliana, maize, rice, soybean, tomato and cotton) and ligneous (poplar) higher plant species (45, 93, 121, 224, 235, 239, 252, 334). Thorough sequences analyses have also been performed in other plant species of agronomical interest such as wheat (76) or grapevine (265)

142 These studies have revealed a great diversity of aquaporins in higher plants, with more than 30 isoforms in all examined species (TABLE 1). Due to a higher degree of ploidy, the genomes of soybean and upland cotton even encode 66 and 71 homologues, respectively. Higher plant aquaporins fall into five subfamilies. Three of these, the Plasma membrane Intrinsic Proteins (PIPs), the Tonoplast Intrinsic Proteins (TIPs) and the Nodulin26-like Intrinsic Proteins (NIPs) are now well-described, with respect to protein localization and function. Two additional subfamilies, the Small basic Intrinsic Proteins (SIPs) (120) and the uncategorized (X) Intrinsic Proteins (XIPs) (59) were discovered more recently. The latter are absent in some higher plant species such as the monocots or the Brassicaceae (FIGURE 2).

151 Genomic studies have also provided insights into the aquaporin family in Algae or early branched land plants (1, 7, 8, 59) (TABLE 1). With respect to seed plants, mosses (e.g. Physcomitrella patens) have two additional subfamilies, the Hybrid Intrinsic Proteins (HIPs) and GlpF-like Intrinsic Proteins (GIPs) whereas spike mosses (e.g. Selaginella moellendorffii) only have HIPs, in addition to the PIPs, TIPs, NIPs, SIPs and XIPs (8, 59). Algae (e.g. Chlamydomonas reinhardtii, Chlorella, Ostreococcus tauri) reside upstream in the plant lineage. They also have PIP and GIP homologues in addition to 5 subclasses (MIP A-E) that, by contrast, seem largely unrelated to other plant aquaporin subclasses (7). Whereas animal and bacterial MIPs fall into both the water channel (aquaporin) and aquaglyceroporin clades, all plant subfamilies except GIPs belong to the first clade (1).

### II.B - Plant aquaporin evolution

#### 1. Interspecific variations

162 Phylogenetic analyses have drawn a general scheme of plant aquaporin evolution. The GIPs may originate by horizontal gene transfer from an ancestral bacterial gene (94) and together with the PIPs have been transmitted from algae to land plants. Whereas XIPs and SIPs likely trace back to early plant (algae) ancestors, other subclasses (HIPs, TIPs) seem to have emerged during land plant evolution, possibly from a PIP ancestor. Interestingly, plant TIPs and mammalian AQP8 share sequence homology and it is as yet unclear whether this reflects a distant evolutionary relationship (1). The origin of NIPs is also uncertain. They may derive by horizontal gene transfer from an ancestral bacterial gene encoding an aquaglyceroporin with solute transport functions. Alternatively,
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NIPs may reflect an evolutionary convergence toward this subclass of aquaporins (1, 330). Finally, some subclasses (such as XIPs, HIPs, or GIPs) were lost during evolution of certain plant lineages pointing to functional redundancies.

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The genetic diversity of aquaporins in plants also reflects the great dynamics of their genomes. While some subfamilies were lost in certain plant lineages (e.g. the XIPs in monocots), key genomic rearrangements have shaped the general expansion of the plant aquaporin family. These events have occurred at different stages during plant evolution. The subdivision of PIPs in PIP1s and PIP2s, or of TIPs in 5 subfamilies may have occurred early since they are conserved throughout all higher plants (FIGURE 2). In contrast, the XIP subfamily may have evolved later as it shows taxon-specific clade divergences (162). In poplar (52), 48 out of 54 aquaporin genes belong to pairs of genes. At variance to what is found in vertebrates (1), most of these pairs (17) are due to genomic duplication whereas a few of them (3) appeared through tandem gene duplications.

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Regarding regulation of MIP expression, some of duplicated genes (principally TIPs) present in poplar showed a significant coregulation whereas others (PIPs) mostly showed divergent gene regulation (52). A precise analysis of tissue-specific expression of the 35 aquaporins in *Arabidopsis* showed that, in this species, there is no strict overlap in expression between isoforms (1), pointing to quick genetic divergence. Evidence of balancing selection has even been provided in PIPs of five tropical trees species, providing a putative response to variable environmental conditions (11).

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## 2. Intraspecific variations

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Plant aquaporin evolution is still at work as indicated by intraspecific variations. In particular, marked differences in aquaporin gene expression between rice cultivars or *Arabidopsis* natural accessions exist under normal or water stress conditions (4, 152, 275). In rice cultivars, differences in aquaporin gene regulation seem to underlie distinct adaptive mechanisms to water deficit (152). The natural variation of plant aquaporin expression and function provides a promising avenue for exploring their integrated function. For instance, genome wide association studies have pointed to the putative contribution of OsTIP3;1 to grain width in rice (109) or to the role of AtTIP2;1 in adaptation of wild *Arabidopsis* to local habitats (77).

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# III) MOLECULAR FUNCTIONS OF PLANT AQUAPORINS

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## III.A - Functional expression and selectivity

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### 1. Expression systems

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*Xenopus* oocytes have been the first expression system used to investigate plant aquaporin function (128, 187). These huge cells are prone to intracellular micro-injection of complementary RNAs and allow simple transport assays based on measurements of cell volume, intracellular pH, or content in radiolabelled molecules. The oocyte system allows addressing transport by aquaporins of water and many other substrates such as organic compounds (glycerol, urea), metalloids (B, Si) or even dissolved gases [CO₂, ammonia (NH₃)]. The oocyte system is still frequently used but displays some disadvantages. Although some intracellular plant aquaporins such as TIPs can be efficiently expressed at the cell surface, some other plant aquaporin subclasses (e.g. PIP1s, SIPs) seem to be recalcitrant to functional expression. In addition, an accurate quantification of proteins present at the plasma membrane remains difficult, restricting the quantitative resolution of transport measurements.

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Yeast cells provide an alternative and have been successfully used for characterization of all 5 plant aquaporin sub-classes. Growth assays can easily be performed, revealing how expression of an
212 aquaporin can enhance the assimilation of some compounds (urea, NH₃) or the toxicity of others.
213 Toxic compounds include hydrogen peroxide (H₂O₂) (29), germanium dioxide (GeO₂) used as a toxic
214 analogue of Si (17), boric acid [B(OH)₃], arsenous acid [As(OH)₃] and antimonite [Sb(OH)₃] (31).
215 However, growth assays provide a rather indirect evidence for transport. Genuine transport
216 measurements on whole cells or spheroblasts are more difficult to develop, but were applied to CO₂
217 (221), ROS (29), NH₃ (22) or selenous acid [H₂SeO₃] (337). Even more challenging is functional
218 reconstitution of recombinant aquaporins in proteoliposomes. This approach was used to dissect the
219 transport activities of PIPs, NIPs and SIPs for water (60, 214, 304), glycerol (60) and NH₃ (112).
220 Reconstitution of aquaporins in gas-tight triblock-copolymer membranes is particularly efficient to
221 detect their CO₂ transport activity (294).

222 **<u>2. Selectivity in 5 sub-classes</u>**

223 All expression systems have revealed that plant aquaporins are multifunctional channels, with a wide
224 range of selectivity profiles (TABLE 2). Most of PIPs and TIPs function as efficient water channels with
225 additional substrates such as H₂O₂ (28, 105) and CO₂ for PIPs, or NH₃ (115, 164) and urea (86) for TIPs.
226 Glycerol has also been investigated as a test solute, by reference to studies in animal and microbial
227 aquaporins (25, 86). Yet, the physiological significance of this transport in plants, for osmotic
228 tolerance in particular, remains uncertain.

229 By contrast to PIPs and TIPs, all NIPs investigated showed at most a reduced water transport
230 activity. NIPs are mostly permeable to small organic solutes and mineral nutrients (169, 278). In
231 particular, they mediate the transport of beneficial [B, Si, selenium (Se)] or toxic [arsenic (As),
232 antimony (Sb)] metalloids (30, 337).

233 Although SIPs potentially display an original pore conformation (see III.A-3) (120, 214), their
234 functional characterization has only revealed a moderate water transport activity. In contrast, XIPs
235 appear as multifunctional channels (26, 162) permeable to water, metalloids and ROS.

236 Whereas mammalian AQP6 is truly permeable to ions (113), no such transport function could
237 be established for plant aquaporins. The electrical conductance induced upon reconstitution of a
238 soybean NIP (Nodulin-26) in lipid bilayers was likely due to protein aggregation artefacts (315) and
239 could not be reproduced in other expression systems.

240 **<u>3. Overall molecular organization and pore conformations</u>**

241 Whereas aquaporin structures have been mostly investigated in animal or microbial homologues, a
242 handful of studies have established crucial structural features of plant aquaporins. Cryo-electron
243 microscopy of PIPs (139) and TIPs (57) confirmed their overall organization as tetramers. More
244 importantly, X-ray crystallography of spinach SoPIP2;1 revealed with an unprecedented resolution
245 the structure of an aquaporin in its closed or open states (217, 286), shedding light onto the original
246 gating properties of PIPs.

247 These data and information deduced by homology modelling have established that plant
248 aquaporins are formed by an assembly of four monomers, each with 6 transmembrane spanning
249 domains (1-6) and five connecting loops (A-E) localized on the intra- (B, D) or extra- (A, C, E) cytosolic
250 sides of the membrane (FIGURE 3; FIGURE 4). Each monomer is able to form an individual
251 transmembrane pore, and remarkably, the A and D loops, which both carry a NPA motif, fold as half-
252 membrane-spanning α-helices and dip into the membrane to position each of their NPA motif at the
253 center of the pore. These two motifs contribute to a central pore constriction and, in conjunction
254 with the dipole moment of the two half-membrane-spanning α-helices, prevent proton (H⁺)
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permeation. Furthermore, four conserved residues form, close to the extra-cytosolic mouth of the pore, a typical aromatic/arginine (Ar/R) constriction functioning as the main selectivity filter.

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Whereas functional selectivity assays have been restricted to a few representative plant isoforms, homology modelling was used to explore in a broader sense the transport selectivity of plant aquaporins, with a main emphasis on the Ar/R selectivity filter (16, 309). Each aquaporin subfamily was found to show specific pore profile(s), and could be further refined in sub-classes with distinct predicted selectivities. For instance, three putative selectivity sub-classes can be identified in NIPs (194, 242, 308). A critical residue of transmembrane spanning domain 2 differs between sub-classes I and II. Reverse substitution of this residue in a class I (Nodulin26) and class II (AtNIP6;1) homolog showed how, in class I, a Ala residue enhances permeation of large solutes at the expense of water transport, whereas in class II, a Trp residue has opposite effects (308). Class III NIPs have small size residues in the Ar/R selectivity filter allowing the passage of large diameter solutes such as silicic acid (194, 242). TIPs also display a great diversity of substrates and Ar/R selectivity filter configurations. A Val-to-Ile substitution at a critical position in loop E was shown to determine the range of transported solutes (14). In barley PIPs, a Ile residue at the end of loop E is also crucial for transport and would confer CO₂ permeability through interaction with a Leu residue in loop C (203). There are also concerns that, in addition to the four individual monomer pores, a fifth pore may be formed at the center of the aquaporin tetramer. The differential inhibition by mercury (see III.B-2) of water and NH₃ transport in a wheat TIP was interpreted to mean that the preferential permeation pathways for these two substrates would be the four monomer pores and the central pore, respectively (22). In X ray structures, the central region of spinach SoPIP2;1 tetramers can accommodate a detergent molecule instead of a lipid indicating that CO₂ permeation through this pathway may be possible, at least from a steric point of view (78).

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### III.B - Gating

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#### 1. Molecular mechanisms

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In vitro studies on purified plant membranes or proteoliposomes have demonstrated the role of cations, H⁺ and phosphorylation in controlling plant aquaporin activity (85, 90, 304). In the case of PIPs, a unifying model of pore opening and closing (gating) was proposed, based on SoPIP2;1 molecular structure (286) (FIGURE 3). This model indicates how the reversible motion of a hydrophobic residue (Leu197 in SoPIP2;1) into and out of the cytoplasmic opening of the pore controls the aquaporin water permeability. This hydrophobic gating residue is carried by the second cytosolic loop (D), the conformational changes of which are mediated by ionic and H-bond interactions with the N-terminal tail (Asp28 and Glu31) and first cytosolic loop (B; Ser 115 and Arg118).

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The pH-dependent gating of PIPs primary relies on protonation of a perfectly conserved His residue of loop D (His193 in SoPIP2;1) (287) (FIGURE 3). At acidic pH, charged His193 interacts with Asp28, Glu31 and loop B (Ser115) to stabilize loop D in a closed pore conformation (79, 286). There also are reports of H⁺-dependent inhibition of TIPs (146, 272), which however involves a His residue located in the second extra-cytoplasmic (intra-vacuolar) loop C. The structural basis of this gating mechanism is as yet unknown.

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Similar to H⁺-dependent gating, the inhibition of PIP water transport by divalent cations can be explained by a direct binding of the cation, which in turn mediates H-bonding between the N-terminus (Gly30 and Asp31) and loop D through loop B, thereby stabilizing the closed pore conformation (FIGURE 3). Although this mechanism was revealed by Cd²⁺ binding in the crystal
structure of SoPIP2;1, the binding site may rather be occupied by Ca²⁺ in vivo. In agreement with the biphasic dose-dependent effects of Ca²⁺ on the water permeability of certain plant plasma membranes (6), a second binding site was recently identified between the loop D and C-terminus of SoPIP2;1 (78). This second binding site may reflect a stabilizing role of the C-terminus on loop D folding.

Structure-function analyses of TIPs, PIPs and NIPs in Xenopus oocytes (90, 122, 186) have pointed to the role of several cytosol-exposed phosphorylation sites in controlling water transport. Structural models of PIPs now support this view indicating how phosphorylation of a conserved Ser residue in loop B (Ser115 in SoPIP2;1) would destabilize the loop D-loop B anchor, thereby favoring the open-pore conformation. Interestingly, phosphorylation of Ser274 on SoPIP2;1 C-terminal tail would act similarly but through a transactivation process, whereby interaction between the C-terminal tail and the loop D of an adjacent monomer would be destabilized after Ser274 phosphorylation.

Due to these great advances, PIP gating is emerging as a paradigm in the aquaporin field. Although distinct protein domains may be involved, similar mechanisms seem to be at work in other plant, animal or microbial aquaporins. For instance, the gating of Arabidopsis NIP7;1 is largely controlled by a unique Tyr residue (Tyr81), located in transmembrane spanning domain 2 and interacting with neighboring Arg220 of the Ar/R selectivity filter to stabilize closed channel conformation (148). In mammalian AQP0 and bacterial AQPz, the corresponding Arg189 residue was shown to adopt distinct conformational states leading to channel gating (102).

Despite recent progresses, a refined structural model of plant aquaporin gating is still needed. For instance, SoPIP2;1 mutants carrying phosphomimetic mutations at Ser115 and Ser274 did not show enhanced water transport activity after reconstitution in proteoliposomes and, accordingly, their X-ray structures displayed a closed-conformation (217). Thus, these mutations may not be sufficient to stabilize the open pore conformation *in vitro* whereas they have an activating role *in vivo* (232). Also, a control of aquaporin activity by osmotic or hydrostatic pressures has been reported in some plant cells or purified membranes (206, 212, 310, 327). It is not yet clear whether these effects are mediated through solute- or pressure-induced changes in aquaporin conformation or through membrane associated cell signaling events (327). Finally, the role for membrane lipids in aquaporin gating is emerging in animals but has not yet been explored in plants (284, 285). In particular, its significance with respect to protein partitioning in membrane micro-domains and stress-induced changes in lipid composition will have to be carefully examined (see IV.B-2).

## 2. Mercury and other aquaporin blockers

Mercury is the most common aquaporin blocker used in plants and animals. It is thought to inhibit the water permeability of purified membranes, cells or tissues, through binding to the thiol groups of crucial aquaporin Cys residues. Mercury may also target His residues as recently revealed in rice OsNIP3;3 (130). The use of mercury must be considered with care, because of its strong cellular toxicity (see VI.B-1) (175, 336). The generality of its inhibiting effects has also been questioned. Firstly, some plant aquaporins are insensitive to mercury because they do not harbor the critical Cys residues identified in other isoforms (58). In addition, mercury did not block but rather activated SoPIP2;1 in proteoliposomes (78). This activation, which was Cys-independent, may reflect a mechanical gating through effects on the lipid environment. In plants as in other organisms (328), there is therefore a strong need for new aquaporin blockers with reduced cellular toxicity. Weak acids or respiration inhibitors (azide, cyanide) decrease cytosolic pH thereby inducing a H⁺-dependent PIP closure. Although they can potentially confirm mercury inhibition (275), these treatments are
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also toxic. Silver and gold compounds offer an interesting alternative (213) but proper application to living cells and tissues seems to be challenging.

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### III.C - Posttranslational modifications

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Although their high hydrophobicity hinders classical biochemical analyses, plant aquaporins are, together with H⁺-ATPases, the most abundant intrinsic proteins of plant plasma and vacuolar membranes. Aquaporins have therefore been prone to extensive proteomic characterizations. In depth mass spectrometry analyses have revealed that plant aquaporins carry numerous co- and post-translational modifications, including phosphorylation, methylation, deamidation, N-terminal acetylation and ubiquitination (44, 62, 132, 216, 254, 255, 297) (FIGURE 4). The latter modification and N-glycosylation were also revealed using immunodetection techniques (143, 303). Thus, the molecular diversity of plant aquaporins goes far beyond a high number of isoforms. The multiple posttranslational modifications of aquaporins point to a variety of regulatory mechanisms targeting aquaporin expression and function. Quantitative proteomics is now revealing how these modifications can vary in abundance, depending on plant tissues or physiological contexts (62, 232). Whereas a general role of phosphorylation in aquaporin gating and trafficking (see IV.B-2) is now well established, the significance of other aquaporin posttranslational modifications is still elusive. Ubiquitination of *Arabidopsis* AtPIP2;1 and N-acetylation of ice plant (*Mesembryanthemum crystallinum*) McTIP1;2 seem to act on endoplasmic reticulum (ER) degradation and redistribution to endosomal compartments, respectively (143, 303). AtPIP2;1 was the first plant membrane proteins and first aquaporin shown to be methylated. Two adjacent residues, Lys3 and Asp6, serve as methylation sites on its cytosolic N-terminal tail (254). These residues overlap with a diacidic motif involved in ER export of the protein (see IV.B-1), suggesting a role for AtPIP2;1 methylation in protein sub-cellular trafficking. Yet, any mutation at these sites had dominating effect on aquaporin trafficking, thereby preventing proper structure function analyses (254). Although deamidation of PIPs shows multiple stimulus-induced changes in *Arabidopsis* roots, the mechanisms leading to this modification and its role are as yet unknown (62).

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### III.D - Heteromerization

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#### 1. Oligomeric structures

Due to their high structural similarity, members of a same plant aquaporin subclass may physically assemble as heterotetramers, thereby enabling multiple molecular and functional combinations. Although their existence remains to be formally demonstrated, heteromers formed of either PIPs or TIPs have been suggested from functional co-expression in *Xenopus* oocytes or yeast, or protein-protein interaction assays such as bimolecular fluorescence complementation (72, 209, 282, 331). Furthermore, molecular modelling of PIPs pointed to a critical role of first extra-cytosolic loop (loop A) in oligomer interactions. This loop contributes to a disulfide bond that stabilizes PIP dimers, which in turn may associate as tetramers (27). Accordingly, site-directed mutations within this loop were able to modify the interaction properties of *Beta vulgaris* (common beet) PIP1s and PIP2s (124).

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#### 2. Functional effects

In plants, heteromerization may be critical for combining PIP1s and PIP2s with distinct functional properties (221). Co-expression studies in *Xenopus* oocytes and maize protoplasts have revealed a clear incidence on trafficking. Whereas singly expressed PIP1s were retained intracellularly, interactions with PIP2s allowed them to reach the plasma membrane (72, 331). Converse effects,
whereby PIP1s may alter the overall tetramer pH sensitivity (20) and transactivate their interacting
PIP2 partner (324) have also been revealed using oocyte expression. Functional expression in yeast
showed that heteromeric assembly of PIP isoforms with a preferential role in water or gas (CO₂)
transport may also provide means for adjusting the overall PIP tetramer selectivity (221). Whereas
these studies point to very complex combinatorial regulations, their significance in the whole plant
remains difficult to assess. For instance, overexpression in transgenic *Arabidopsis* of a PIP2 mutant
form deficient in trafficking to the plasma membrane induced an intracellular retention of native PIPs
(and a decrease in root water transport) (270). Although this was not strictly demonstrated, these
effects may be due molecular interactions between PIPs in the plant.

### III.E - Perspectives

The present section shows that we have got a fair understanding of plant aquaporin molecular
functions. This knowledge is central for addressing aquaporin function and regulations at the plant
cell and whole organism level. A powerful, but challenging integrative approach is expression in
transgenic plants of site-directed mutant forms of aquaporins. The chapters below exemplify how
aquaporin phosphorylation and trafficking are now analyzed along these lines. By comparison, in
*planta* analysis of aquaporin properties such as transport specificity or H⁺- or Ca²⁺-dependent gating
is lagging behind. Addressing the functions and regulations emerging from aquaporin heteromeric
assembly also represents an important but highly challenging objective.

## IV) CELL BIOLOGY OF PLANT AQUAPORINS

### IV.A - Subcellular localization patterns

In relation to their unique metabolic capacities (e.g. photosynthesis, N₂-fixation through
endosymbiosis), plant cells are characterized by a great diversity of intracellular compartments and
subcellular membranes. Accordingly, plant cells display a wide palette of aquaporin subcellular
localization patterns.

#### 1. Plasma membrane

As in animals, the plant plasma membrane represents a crucial barrier and exchange platform.
Accordingly, this membrane harbors three subclasses of aquaporins, namely the PIPs (FIGURE 5A),
NIPs and XIPs (26). Fusions with fluorescent protein reporters have indicated that most of these
aquaporins are expressed on the entire cell surface. Yet, some isoforms can be confined to
membrane sub-domains. For instance, two NIP homologs, *Arabidopsis* AtNIP5;1 (277) and rice Lsi1
(OsNIP2;1) (277), are specifically expressed on the endofacial side of root endodermal cells, to
mediate a passive cellular efflux of B and Si, respectively. Transporters specialized in the cellular
influx of the same solutes (BOR1 and Lsi2, respectively) show an opposite polar localization. Thus,
these complementary arrangements create an efficient path for transcellular transport of B and Si,
and centripetal transfer into vascular tissues (xylem vessels).

A few PIP isoforms also show a somewhat polar expression, with preferential accumulation on
the exofacial and endofacial sides of root cells in rice and maize, respectively (95, 250). The PIP
isoform counterparts that must be expressed on the opposite cell side to mediate transcellular water
transport remain to be identified.

Recent imaging of PIPs using Fluorescence Recovery After Photobleaching (FRAP) or
evanescent wave microscopy techniques also revealed their extremely low lateral mobility at the cell
427 surface (107, 150, 167, 181). This feature may partly result from hindering interactions with the cell
428 wall (181). Single particle tracking further showed a confinement of PIPs in membrane micro-
429 domains associated to specific sterol compositions (150). Accordingly, PIPs were found to be
430 enriched in detergent-resistant membrane fractions tentatively associated to membrane lipid rafts
431 (131, 199, 202). This membrane partitioning likely underlies the dynamics of PIP cycling between the
432 cell surface and endosomal compartment (see IV.B-2).

433 **2. Vacuoles**

434 Vacuoles represent the most voluminous intracellular organelles of plant cells. Vacuoles are not
435 uniform, with distinct subtypes present in a same cell and showing specialized lytic or protein storage
436 functions. Although vacuoles harbor a single subclass of aquaporins, the so-called TIPs (319) (FIGURE
437 5B), each vacuolar subtype is characterized by a specific set of isoforms (118). It has been argued that
438 these patterns must be determined by targeting pathways specific of each vacuolar subtype.
439 However, they are more likely generated through one or several common trafficking machinery(ies)
440 and simply result from distinct developmental expression of TIP isoforms during the course of cell
441 differentiation (111, 118). The vacuolar membrane (tonoplast) is more fluid than the plasma
442 membrane and, by comparison to PIPs, TIPs show a much higher (> 10-fold) lateral membrane
443 mobility (107, 167). Yet, their expression is not uniform and preferential expression of TIP fused to
444 GFP has been observed in intra-vacuolar invaginations (bulbs) and in apposing tonoplast regions of
445 two adjacent vacuoles (19, 248). This pattern may allow privileged exchanges between neighboring
446 vacuoles. It may also partly result from antiparallel dimerization of GFP expressed in two apposing
447 membranes (262).

448 **3. Other intracellular compartments**

449 Whereas most plant aquaporins can be observed in the ER, during biogenesis and transfer to their
450 destination membrane, some isoforms such as SIPs (114, 214) and some NIPs (197) seem to be
451 resident of this compartment. Their mode of targeting and cellular function in this compartment are
452 as yet unknown.

453 The chloroplast is a plant specific organelle, delineated by an envelope formed by a double
454 membrane. The chloroplast also contains numerous stacks of thylakoid membranes, which harbor
455 the light-collecting antenna and electron transfer chains crucial for photosynthesis. Thorough
456 proteomic analyses in *Arabidopsis* (70, 71) have suggested the presence of PIPs and TIPs, in the inner
457 envelope and thylakoids, respectively. Localization of a PIP1 (*NtAQP1*) in the chloroplast envelope of
458 tobacco leaves has also been revealed by immuno-cytochemistry (295). Whereas these localizations
459 point to highly relevant plant specific function (see V.D), they now call for complementary data and
460 confirmation in other plant materials.

461 Mammalian AQP8, which shows a somewhat atypical sequence among other animal
462 aquaporins and significant homology to TIPs, has been localized to mitochondria (38). A putative
463 mitochondrial targeting signal was also detected in pollen-specific *Arabidopsis* *AtTIP5*;1 (272). Ectopic
464 expression in the vegetative cell of transgenic pollen of *AtTIP5*;1 fused to a fluorescent reporter led
465 to the proposal that *AtTIP5*;1 localizes to mitochondria. Yet, more adequate reporter fusion and
466 colocalization studies revealed that this conclusion is erroneous and that *AtTIP5*;1 actually localizes
467 to the tiny vacuoles of pollen sperm cells (320). Thus, as far as we know, plant mitochondria seem to
468 be deprived of aquaporins.
4. Dual localization patterns
---

469
470 It is assumed that the high degree of cell compartmentalization in plants has represented a major
driving force during the molecular and functional diversification of aquaporins in these organisms. In
471
472 summary, plant aquaporins have been localized throughout the cell secretory system including ER,
473 Golgi, endosomes, autophagosomes and vacuoles (98, 168). Specific isoforms are also expressed in
474 chloroplasts and in the symbiosome membrane that surrounds, in legumes, the intracellular N₂-fixing
475 bacteroids of Rhizobiaceae-infected roots (see VIII.A). In contrast, and for some unknown reasons,
476 aquaporins seem to be excluded from some compartments such as plant mitochondria and
477 peroxysomes.
478

478
479 Interestingly, examples of dual subcellular localizations of aquaporins have recently emerged
in plants. For instance, the PIP1 homolog, *NtAQP1*, is expressed in the plasma membrane and
480
481 chloroplast of tobacco leaves (295) while TIP3s have been localized in both the plasma membrane
482 and tonoplast of maturating *Arabidopsis* seeds (82). A vacuolar TIP1 homolog can also be transiently
483 expressed on the symbiosome membrane of *Medicago truncatula* (barrel clover) root nodules (84).
484 As discussed below, these patterns must be determined by exquisite trafficking signals, which most
485 often remain to be identified.
486

IV.B - Aquaporin trafficking
---

1. Multiple pathways in the secretory system
***

487
488 The modes of plant aquaporin targeting to their destination membranes have been addressed using
various approaches. Firstly, site-directed mutations of maize and *Arabidopsis* PIP2s revealed that
489
490 inner sequence motifs direct their trafficking to the plasma membrane. In particular, diacidic motifs
491 located on their N-terminal tail likely interact with the COPII sorting machinery and critically
492 determine their export from the ER (270, 332) (FIGURE 6A). Anterograde transport of PIP2s in the
493 secretory pathway also involves a LxxxA motif (48) in the third membrane spanning helix and a
494 phosphorylation site located on the C-terminal tail (234). Most PIP1s do not have these motifs but
495 are likely dragged to the plasma membrane through heteromer assembly with PIP2s.
496

497
498 The motifs that determine TIP routing to the vacuole remain largely unknown. A
pharmacological screening in *Arabidopsis* (241) revealed that TIP1 targeting is sensitive to Brefeldin A
499
500 and likely involves a Golgi-dependent route (FIGURE 6A). By contrast, trafficking of TIP2s and TIP3s
does not pass through the same route and is sensitive to a newly identified compound named C834
501
502 (241). In the future, a chemical genetic approach may be used to identify the molecular components
503 of these different pathways.
504

505
506 Q-SNAREs of the syntaxin family are well identified molecular players of vesicular trafficking in
507 eukaryotes. In plants, they mediate the trafficking of PIPs and TIPs at specific stages along the
508 secretory pathway (24, 291). In particular, post-Golgi trafficking of *Arabidopsis* AtPIP2;7 was shown
509 to depend on a direct physical interaction with two specific syntaxins, SYP61 and SYP121 (96).
510

2. Constitutive cycling of PIPs and response to environmental stresses
---

505
506 Although they have a reduced lateral mobility, PIPs are far from static at the plasma membrane.
507
508 Pharmacological and genetic studies have revealed that they continuously cycle between the plasma
509 membrane and early endosomes (Trans-Golgi Network) (167) (FIGURE 6A). Under resting conditions,
510 this process is mediated through clathrin-mediated endocytosis (61) and is blocked in part by the
511 auxin analogue, NAA, using an as yet unknown mechanism. In root epidermal cells under osmotic or
512 salt stress, the cycling rate of PIPs is enhanced due in part to the activation of a complementary
512 endocytic pathway, independent of clathrin, and associated to membrane micro-domains (150, 167)
513 (FIGURE 6B). These processes are probably linked to the enhanced mobility of PIP molecules at the
514 cell surface observed under osmotic and salt stress conditions (107, 150).

515 In connection to these processes, the same stresses induce a partial internalization of PIPs (34,
516 35) (FIGURE 6B). This leads to a reduced abundance of PIPs at the root cell surface, which may
517 contribute to the observed decrease of root water permeability. In addition, osmotic stresses induce
518 functional and morphological rearrangements of the vacuolar apparatus. For instance, they trigger a
519 relocalization of TIPs, from the vacuole to endosomal compartments or intravacuolar bulbs, in ice
520 plant and *Arabidopsis*, respectively (34, 303).

521 These profound effects of stresses on aquaporin sub-cellular localization and dynamics are
522 central in plant responses to their environment. Beyond their impact on plant water relations, they
523 represent one of earliest cellular responses to stresses and can possibly be extended to other
524 membrane proteins. In addition, stimulus-induced PIP trafficking can be antagonized by ROS
525 scavengers, in agreement with the central role played by ROS in stress and hormonal signaling in
526 plants (35) (FIGURE 6B). Deciphering the molecular and cellular mechanisms that govern PIP and TIP
527 dynamics may therefore be central to understand the perception and transduction of stress signals in
528 plants. In *Arabidopsis* roots, both salt and oxidative stresses induce quantitative changes in the
529 double C-terminal phosphorylation of AtPIP2;1 (62, 234). Phosphorylation of a specific residue
530 (Ser283) interferes with trafficking of internalized PIPs to fuzzy structures or spherical bodies. These
531 structures have tentatively been identified as sorting endosomes and prevacuolar compartment
532 (multivesicular bodies), respectively (234) (FIGURE 6B). Whether this sorting permits a reversible
533 sequestration of PIPs, prior to remobilization to the plasma membrane, or whether it directs them
534 towards vacuolar degradation is unknown. Nevertheless, these mechanisms are reminiscent of the
535 vasopressin- and phosphorylation-dependent trafficking of Aquaporin-2 in renal epithelial cells (198).

### 3. Degradation pathways

536 Although aquaporin abundance can markedly vary in response to abiotic stresses or oscillate along
537 circadian rhythms, information on the modes of plant aquaporin degradation has remained scarce. A
538 RING membrane-anchor E3 ubiquitin ligase from pepper, and its *Arabidopsis* homologs, were shown
539 to localize in the ER and ubiquitinate AtPIP2;1 leading to its retention in this compartment (143). This
540 process was enhanced under osmotic stress, leading to aquaporin degradation through the
541 proteasome pathway (FIGURE 6C). A more recent study established a very novel link between
542 aquaporin degradation and intracellular trafficking in *Arabidopsis* (98). AtPIP2;7 was shown to
543 interact in the ER and Golgi, with a membrane protein, named TSPO for tryptophan-rich sensory
544 protein/translocator and serving as multistress regulator (FIGURE 6C). The complex was then
545 directed towards vacuolar degradation, using the autophagosome pathway, this process being
546 stimulated by ABA. Thus, plant cells under water stress can use various pathways, for aquaporin
547 degradation and long term down-regulation of plasma membrane water permeability.
548

### IV.C - Perspectives

549 The present section showed that, in addition to multiple substrates and selectivity profiles, plant
550 aquaporins show very diverse subcellular localization patterns, and cell responses to hormonal and
551 environmental stimuli. These properties can provide a first hint at their high genetic diversity. We
552 also note that aquaporins have initially been used as mere reference markers for the plasma
553 membrane or the vacuole. An accumulating body of data now reveals their remarkable trafficking
554
555
556

properties. Aquaporins are therefore emerging as one of major membrane protein models in plant cell biology (168).

557 While evanescent wave fluorescent microscopy and associated single particle tracking techniques provide novel insights into plant aquaporin dynamics, much remains to be learned about the molecular and cellular mechanisms that direct their trafficking, both under resting and stress conditions. The recent characterization of individual aquaporin partners has provided insights into specific cell sorting or degradation steps (96, 98, 143) (TABLE 3). Genomic studies based on classical (53) or split-ubiquitin (123, 140) yeast two-hybrid systems are now revealing a myriad of additional aquaporin partners (FIGURE 7). These genome-wide aquaporin interactomes and their functional genomic characterization will undoubtedly reveal novel mechanisms related to aquaporin trafficking. It should also reveal hormonal and environmental signaling chains acting on aquaporins or large protein complexes coupling water transport to other membrane processes (TABLE 3).

567

# V) CELL-AUTONOMOUS AND INTRACELLULAR FUNCTIONS

568

## V.A - Osmoregulation

569
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Addressing the cell-autonomous functions of aquaporins provides a useful step towards integration of their role at the organ and whole plant levels. One major cellular function of aquaporins is osmoregulation.

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In agreement with the targeting of specific aquaporin isoforms to distinct subcellular compartments, water transport assays in purified membrane vesicles, isolated vacuoles and protoplasts have revealed that membranes from a same cell can exhibit strikingly different permeability profiles. In tobacco cells for instance, purified tonoplast vesicles were 100-fold more permeable to water than their plasma membrane counterparts (188). An extremely high water permeability in the tonoplast seems to be common to most of plant cells (188, 204, 212). According to mathematical models, this property would provide the cell with the capacity of reducing peak fluctuations of cytosolic volume in case of a sudden change in extracellular water potential (208, 290). Thus, plant cells use their large vacuoles as buffering compartments during cell osmoregulation.

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The delayed volume response of isolated protoplasts subjected to an osmotic challenge indicated that dynamic changes in water permeability may also contribute to plant cell responses to osmotic stresses (206). A direct dependence of water permeability on transmembrane osmotic gradients has been observed in tonoplast vesicles purified from wheat roots, but not in tobacco suspension cells (188, 212). Although the mechanisms leading to rapid and possibly direct changes in aquaporin activity have not yet been addressed in these contexts, we assume that they pertain to the general gating and trafficking properties described in the two previous sections (see III and IV).

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595

The dynamic responses of aquaporins to osmotic stress have led to the idea that aquaporins, localized on the vacuolar or plasma membrane may serve as osmosensors, in guard cells during stomatal movements or in growing pollen tubes for instance (173, 263). These ideas remain highly speculative and rely at most on experiments showing that osmoregulation is altered by mercury, a non-selective aquaporin inhibitor. More solid genetic evidence is now needed to support such models. Despite the presence of aquaporins in most intracellular compartments, we also ignore the volume dynamics and osmotic or mechanical constraints at work on their membranes during volume or turgor regulation. Finally, most studies have focused on plasma membrane and vacuolar
aquaporins and a closer characterization of SIPs, aquaporins that are confined in the ER, may reveal
novel aspects of plant cell osmoregulation (114, 214).

V.B - ROS detoxification and signaling

Consistent with the ability of TIPs to transport H₂O₂ in yeast (29), transgenic overexpression (312) or inactivation of these aquaporins (258) can confer plant tolerance or sensitivity to oxidative stress, respectively. Among ROS, H₂O₂ has a relatively long life time and its import by TIPs into the vacuole may efficiently contribute to ROS detoxication. This implies, however, that a detoxication machinery is expressed in vacuoles. Up to now, most of these systems have been characterized in the cytoplasm.

PIP2s can also have a significant H₂O₂ transport capacity (66) and the expression of some of these aquaporins is reduced by H₂O₂ itself (105). Considering that H₂O₂ produced in the apoplasm by combined NADPH-oxidase and superoxide dismutase activities can serve as a signaling intermediate, a role of PIPs in plant cell signaling is very likely. Such signaling role, which has been established for mammalian AQP1, AQP3 and AQP8 (28), still awaits experimental demonstration in plants. Guard cells, which respond to ABA through H₂O₂ production, may represent an interesting model to address this issue.

V.C - Vacuolar storage

The permeability of tonoplast aquaporins to compounds such as NH₃ (164) or urea (86, 160) has suggested a role for TIPs in vacuolar storage and remobilization of nitrogen metabolites. However, this role could not be validated *in planta* using reverse genetic approaches (164). In addition, plant membranes are equipped with other transport systems that mediate a more specific and vectorial transport of NH₃/NH₄⁺ and urea. Thus, the role of TIPs in vacuolar partitioning of nitrogen metabolites seems at most minor.

V.D - Chloroplast transport

Because of its roles in cell bioenergetics and photosynthesis, the plant chloroplast is a unique organelle in eukaryotic cells (FIGURE 8). One key reaction is water oxidation to molecular oxygen that occurs in the thylakoid lumen. It was calculated that this consumption of water molecules represents 60- to 170-fold the thylakoid lumen volume per day (18). Although NMR measurements indicate a moderate diffusional water permeability of the thylakoid membrane, a minute osmotic gradient (≤ 3 mOsm) might be sufficient to support the steady uptake of water required for thylakoid lumen refilling (18). Thus, aquaporins may be dispensable during this process. In contrast, the thylakoid lumen shows fast and marked volume increase in response to light, which may be facilitated by aquaporin activity.

CO₂ is another potentially relevant substrate of aquaporins in the chloroplast. Several physical and biochemical components of plant cells, such as cell wall composition and tortuosity, unstirred layers and carbonic anhydrase activities in the vicinity of membranes, or CO₂ permeability of the membranes themselves possibly restrict CO₂ diffusion throughout plant cells (69). The chloroplast envelope may represent a critical barrier, limiting carboxylation reactions performed in the stroma through the Calvin-Benson cycle. Interestingly, the CO₂ permeability of chloroplast envelopes purified from tobacco leaves was 5-fold lower than that of PM vesicles and was reduced by ~ 90% after antisense inhibition of NtAQP1 in transgenic tobacco (295). It is now critically needed that these data are extended to other plant materials.
# VI) WHOLE PLANT WATER TRANSPORT AND TRANSPRATION

## VI.A - Context

## VI.B - Root water transport

### 1. Structure-function analyses
679 to 64% of Lp<sub>r</sub>, respectively (175, 275). At present, plant aquaporin pharmacology remains highly 680 limiting, all inhibitors described being potentially toxic and exerting numerous secondary effects. 681 Nevertheless, a higher reliability can be obtained when inhibitors with different modes of action 682 (mercury vs. weak acids or azide) yield similar estimates of the aquaporin-dependent path (275).

683 Aquaporin expression patterns can also provide useful hints at the contribution of water 684 channels to root water transport. These patterns have been investigated in roots of many plant 685 species with most accurate descriptions in *Arabidopsis* (83, 119, 230) (FIGURE 9B-D) and cereals such 686 as rice (251), maize (97) and barley (136). Besides their agronomical importance, cereals offer an 687 interesting context for studying root function, with seminal and shoot-borne roots showing distinct 688 morphologies and functions. Aquaporin expression studies in roots have focused on PIPs, which 689 supposedly play a crucial role in plasma membrane and transcellular water transport, and TIPs, which 690 show high expression levels in roots. For instance, the *Arabidopsis* root expresses at least 7 PIP and 6 691 TIP isoforms with specific expression patterns, the latter being expressed in all tissues except the 692 vasculature and the root tip meristem (83). In all plant species examined, very precise and distinct 693 cell-specific expression patterns could be established for the numerous aquaporin isoforms present 694 in roots (97, 251). Overall, the data stress the importance of controlling local hydraulic properties all 695 along the radial pathway, with preferential expression of some isoforms in the exodermis or 696 endodermis (97). Apoplastic barriers are formed in these two cell layers, through lignin deposition at 697 Casparian strips and subsequent suberization of the whole apoplasm, thereby creating specific 698 limitation for water transport. In addition, high expression of some other isoforms in the stele is 699 consistent with centripetal transport of water towards the root vasculature, whereby water flows 700 have to be mediated by reduced exchange surfaces (80, 230) (FIGURE 9B-D). Aquaporin expression 701 profiles were also shown to vary along the root axis, indicating that the radial pathway itself varies 702 during root growth and differentiation. In some cases, these studies were coupled to water transport 703 measurements in well-defined root zones (80, 136). Recent comparisons of water transport and 704 aquaporin expression patterns in woody perennial and herbaceous plants have indicated distinct 705 root hydraulic differentiation profiles between these types of plants (80). In grapevine for instance, 706 the radial pathway seems to be extremely tight in differentiated root segments, due to the formation 707 of a periderm, suggesting that soil water is taken up through root tips, mostly.

708 In complement to expression analyses, reverse genetics has brought unequivocal evidence for 709 the contribution of PIPs to Lp<sub>r</sub> (119, 226, 230, 267). Although of modest amplitude (10-20% change in 710 Lp<sub>r</sub>), significant water transport phenotypes could be observed in single knock-out mutants. Yet, 711 these studies have remained restricted to a few isoforms and a genetic redundancy between close 712 homologues has to be considered (226). Thus, we still lack a comprehensive molecular view of root 713 hydraulics, whereby each isoform may be associated to water transport at specific cell sites and 714 under a given type of force.

715 Modelling of root water transport, along concentric cell layers and the root axis, or in distinct 716 root subtypes (37, 137), can provide a significant support to these functional and genetic analyses. 717 These modelling studies indicate that the site of water absorption (root tips vs. whole root) may vary 718 between species. A future challenge will be to integrate root hydraulic functioning in a soil to 719 apprehend all kinetic and spatial refinement of water uptake in a natural environment (64). These 720 studies should not be restricted to herbaceous species. For instance, the use of natural caves 721 revealed that aquaporins of live oak and gum bumelia (*Sideroxylon lanuginosum*) contribute to root 722 water uptake deep into the soil (18-20 m) where they can respond to seasonal and diurnal rhythms 723 (190).
724

## 2. Water stress

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731

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734

735

Direct exposure of roots to water stress usually results in inhibition of aquaporin activity and water transport at the cell and whole organ levels (34, 43, 99, 227, 275). Yet, some plant varieties or natural accessions show opposite responses with an enhancement of root cell hydraulic conductivity (99, 275). The effects of external (106, 176) or endogenous ABA (223) are also contrasting, during time and between species. For instance, ABA transiently enhances cell hydraulic conductivity in maize root cells (106) whereas it inhibits $L_{p_r}$ in aspen (311). These observations highlight the variety of hydraulic strategies in response to soil drying. Whereas increased $L_{p_r}$ during the early phase of drought could help optimize the capture of soil water resources, a long term inhibition provides a more conservative mechanism for the plant, to prevent a reverse flow of water, from the plant root into a dry soil. Regulation of whole root water transport can also have a dramatic hydraulic impact on leaves, to favor or reduce growth or act on stomatal aperture (transpiration) (see VII.A-3).

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743

It is now required that we go beyond classical and abrupt water stress settings used in laboratories. In particular, the heterogeneity of soil water content in natural conditions will have to be considered more carefully in future experimental studies, to understand how plant roots sense soil water deficit and manage soil water on the long term. Partial root-zone drying in a riparian tree (*Melaleuca argentea*) resulted in threefold enhancement of root hydraulic conductance in the wet soil portion, with a slight increase in PIP1 expression, by a signaling mechanism that remains to be identified (191). Also, heterogeneities in local hydraulic conductivities could interfere in water redistribution (hydraulic lift) between soil layers varying in water content (40).

744

## 3. Nutrient availability

745

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747

748

749

750

751

752

Root water transport and aquaporin activities are extremely sensitive to nutrient availability in the soil. Plant deprivation in most macronutrients (phosphorus, nitrogen or sulfur) leads over a couple of days to a decrease in $L_{p_r}$ (42, 62) through aquaporin down-regulation (FIGURE 10). It is assumed that these regulations may favor whole water uptake in conditions favorable to plant growth. Considering possible soil heterogeneities, these regulations may also enhance nutrient drag to the root in soil zones that are the richest in nutrients. At variance with these effects, potassium ($K^+$) starvation enhances $L_{p_r}$ (157, 236). The modes of aquaporin regulation by nutrients seem to be complex since they involve mixed effects on aquaporin expression and phosphorylation (62, 88, 157, 313).

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The signaling mechanisms that mediate these effects are as yet unknown but clearly involve hormone responses. Thus, ethylene was shown to mediate the inhibiting effects of phosphorus starvation on $L_{p_r}$ in *Medicago falcata* (151) whereas ABA enhanced $L_{p_r}$ stimulation by $K^+$ starvation in sunflower (236). It will also be important to explore the respective contributions of local and systemic signals in these regulations (87).

758

## 4. Other soil signals

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760

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762

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764

765

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767

Both low temperatures and oxygen deprivation in the soil exert a general inhibiting effect on root water uptake through decrease of cell and whole root hydraulic conductivities. Cold exerts complex up- and down-regulating effects on PIP expression in roots (144, 329). ROS and calcium, which accumulate in these conditions, and aquaporin phosphorylation may provide complementary mechanisms for cold-induced $L_{p_r}$ regulation (144, 145). In rice, a long-term (2-5 days) exposure of roots to low temperature induced a compensatory increase in $L_{p_r}$, due in large part to enhanced expression of OsPIP2;5 (2). Interestingly, this induction required that the shoot was maintained both at a control temperature and in its integrity, suggesting that a shoot-to-root signal was involved (see VI.B.6).
768

Soil flooding and compaction prevent oxygen diffusion, thereby creating severe anoxic stress, especially onto actively growing root tips. This stress results in a strong metabolic imbalance, with a marked cytosolic acidosis, which in turn mediates H⁺-dependent gating of PIPs (287). The long term effects of anoxia are mediated through a general inhibition of aquaporin gene expression (156). Interestingly, the *AtNIP2;1* gene shows a strong induction in *Arabidopsis* roots under anoxic stress. This aquaporin, which transports lactic acid, may facilitate the leak into the soil of this fermentation product, thereby preventing excessive cell acidification (50).

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## 5. Diurnal and circadian rhythms

776

In relation with diurnal changes in transpiration, many plant species show a peak in $L_{p_r}$ during the day (103, 250). This regulation can be accounted for by an increase in root aquaporin transcripts during the early phase of the day (163, 279, 298) and a slightly delayed peak in protein abundance (99, 250). Experiment under constant light and/or in circadian clock mutants, have shown that at least in maize and *Arabidopsis*, these oscillations are under circadian control (39, 279). In maize, the amplitude of these oscillations is dramatically amplified by previous exposure of plants to water limiting conditions, in the air or in the soil. This climatic control would optimize water uptake in moderate dry soils, by preserving rhizosphere hydration (39). Roots are also able to directly sense light. For instance, the photoreceptor phytochrome A controls the enhancement of AtTIP2;2 expression during adaptation of the *Arabidopsis* root to darkness (296).

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## 6. Transpirational demand and other shoot-to-root signals

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Recent studies in rice and poplar (142, 250) have established a direct role of the transpirational demand in $L_{p_r}$ regulation. This was made possible by independent manipulation of several factors affecting transpiration (i.e. light or relative air humidity) to discard primary effects of these factors on aquaporin functions. In both plant species, transpiration triggered a dramatic increase in PIP gene expression in roots.

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Identifying systemic signals conveyed from the shoots to regulate root hydraulics represents one of most exciting questions in this field. Several studies have identified context to address this topic. For instance, shoot topping in grapevine, soybean and maize was shown to reduce $L_{p_r}$ and root aquaporin activity and expression in a few tens of minutes (299). Whereas auxin or phloem-borne signals seem to be excluded, hydraulic signals transmitted through the xylem are more likely. In aspen (*Populus tremuloides*) seedlings, defoliation results in adjustments of both leaf and root hydraulics. The decrease in $L_{p_r}$ observed after one day was associated to down-regulation of a major PIP1 isoform (159). In wheat, partial root excision did not alter stomatal conductance nor transpiration but increased after 1.5h the hydraulic conductivity of the remaining root by several fold, in relation to enhanced accumulation of ABA in this organ (307).

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# VI.C - Leaf water transport

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## 1. Hydraulic resistances in the leaf

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Following root uptake, the soil water enriched in nutriments is transported as sap to the plant shoot through xylem vessels formed of dead cell structures. Except under severe drought (49), xylem transport in the stem is non-limiting. In contrast, the leaf represents, in addition to the root, a major checkpoint for plant water transport. In fact, the hydraulics of inner leaf tissues is designed for efficient sap delivery through a network of fine xylem vessels down to substomatal chambers, where water evaporates and is transpired through the stomatal pores.

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Leaf hydraulics is therefore determined by vascular (xylem) and extra-vascular resistances, the respective contributions of which vary according to plant species and leaf morphology (233). The extravascular pathway is formed of parenchyma cells packed along the vessels, surrounded by a tight bundle sheath and by the mesophyll, which shows a looser compaction with numerous lacunas (FIGURE 11A). Similar to roots, water can flow in leaves along the apoplastic path or from cell-to-cell.

The contribution of aquaporins to leaf water transport has been dissected using approaches similar to those used in roots. Expression studies revealed a high aquaporin abundance in the elongating zone of cereal leaves, in the vascular bundles of most plant species and to a lesser extent in the mesophyll (23, 95, 232) (FIGURE 11B). Reverse genetic analysis in *Arabidopsis* identified three PIP isoforms that contribute to leaf hydraulics (230, 232). Several lines of evidence indicate that, in *Arabidopsis* at least, the veins (xylem parenchyma and bundle sheath cells) rather than the mesophyll are hydraulically limiting. Firstly, all PIPs contributing to rosette hydraulic conductivity shared a common expression in the vascular bundles (232). Secondly, the water permeability of protoplasts purified from the veins but not from the mesophyll showed a regulation by light and ABA that paralleled that of the whole leaf (232, 264). Thirdly, complementation, using vein-specific expression of AtPIP2;1, of a *Atpip2;1* knock-out mutant lacking light-dependent regulation of leaf hydraulics was sufficient to restore a wild-type response (232).

## 2. Signals

Leaf hydraulics is controlled by numerous environmental or hormonal signals, which most often establish a functional coupling between regulation of inner leaf water transport and stomatal movements. For instance, ABA inhibits inner leaf water transport by down-regulating aquaporin activity in bundle sheath cells (264). These effects are physiologically consistent with an additional and direct induction of stomatal closure by the hormone. More generally, a strong correlation between leaf hydraulic and stomatal conductances and expression of a TIP2 homologue was observed in grapevine leaves under both water sufficient and water stress conditions (231).

Besides water stress, the light regime is the other signal that dominates leaf hydraulic regulation. In most plant species, leaf hydraulic conductance is maximal during day time and a circadian control was established in sunflower (210). In several tree species including walnut (*Juglans regia*), pedunculate oak (*Quercus robur*) and common beech (*Fagus sylvatica*), light-dependent leaf hydraulic conductance showed a good correlation to PIP gene expression (15, 51). In *Arabidopsis*, by contrast, quantitative proteomics revealed that the di-phosphorylated form of AtPIP2;1 was closely linked to changes in rosette hydraulic conductivity under varying light or dark regimes (232). The ability of a phosphomimetic but not of a phosphodeficient form of AtPIP2;1 to restore the light response of a *Atpip2;1* knock-out mutant established that phosphorylation of this single isoform is necessary for light-dependent regulation of leaf hydraulics.

Crassulacean acid metabolism (CAM) allows some plants to adapt to low-water environment by day/night variation in CO₂ uptake and fixation. In leaves of ice plant, the osmotic water permeability of purified protoplasts and expression of three PIPs and a TIP isoforms peaked at the end of the light period, together with accumulation of CAM cycle metabolites. Thus, diurnal regulation of aquaporins may favor cell osmoregulation and leaf water balance throughout the CAM cycle (302).

## 3. Guard cells and leaf movements

Stomatal aperture is constantly adjusted through reversible changes in guard cell volume and plays a major role in controlling plant transpiration. Whereas a role for aquaporins in stomatal movement
has long been hypothesized, supporting evidence has remained scarce, rather suggesting a role in stomatal closure. For instance, over-expression of a *Vicia faba* PIP1 in *Arabidopsis* accelerated the stomatal response to dark and ABA (55). In sunflower guard cells, a TIP isoform showed a diurnal peak in expression that coincides with stomatal closure (256). With regard to the plethora of information on hormone signaling and membrane transport regulation in guard cells, getting a better understanding of aquaporin function in these cells is now becoming critical.

Other types of movements in leaves seem to be associated to circadian regulation of aquaporins. In rain tree (*Samanea saman*), such regulation contributes to circadian variation of osmotic water permeability in motor cells and as a consequence to diurnal leaf movements (205). Similarly, epinastic movement of tobacco leaves is dependent on circadian expression of a PIP1 homologue (NtAQP1) in the petiole (266).

## 4. Embolism repair

Long-distance transport of water in vascular plants can be dramatically impeded by the formation of embolisms in xylem vessels, following tissue freezing during winter (249) or under extreme drought, when intense transpiration and limiting water supply result in high tension within the vessels (165). Changes in aquaporin expression in relation to embolism recovery, and inhibition of this process by mercury have suggested that aquaporin-facilitated water transport can contribute to embolism refilling (165, 249, 260). Accordingly, transgenic poplar and *Arabidopsis* with reduced expression of PIPs were more susceptible to drought-induced embolisms and showed reduced recovery response of whole plant water conductance upon rewatering, respectively (182, 259). Yet, the modes of water and solute co-transport to allow embolism refilling in tissues under strong water tensions are still debated (104, 316).

## 5. Water uptake

In some very specific physiological contexts or species, leaves can substitute for roots in taking up external water. In atmospheric epiphytes for instance, water absorption of air moisture through trichomes can provide a strong adaptation to drought (218). In conifers, leaf absorption of melting snow may support embolism refilling after winter (141). In both cases, a role of aquaporins in water equilibration within leaf tissues was suggested by aquaporin expression patterns or pharmacological inhibition.

## VI.D - Perspectives

### 1. Molecular dissection of plant tissue hydraulics

Despite an expected genetic redundancy within the aquaporin family, a few studies have identified single aquaporin isoforms that, under laboratory conditions, significantly contribute to root or leaf hydraulics (56, 119, 226, 230, 232). These advances will hopefully encourage a more thorough genetic dissection of plant tissue water transport. Also, a special focus should be put onto aquaporin function in cell layers that are thought to be hydraulically limiting, such as the endodermis and exodermis in roots or the bundle sheath cells in leaves. For instance, a crucial role of the aquaporin pathway in suberized root tissues has been hypothesized in most text books but was only investigated in a handful of studies (80, 238). One critical approach in the future may be to use tissue-specific promoters for cell sorting or directed expression of aquaporins (232, 244, 264).

Most importantly, the identification of master regulators of aquaporins in these cell contexts will be critically needed. In the future, quantitative genetics of plant hydraulics in plants under control or stress conditions (275) may provide a privileged access to these regulators. For now, and
as outlined below, the dissection of aquaporin regulation networks using the power of transcriptomic
and proteomic approaches provides more operational strategies.

## 2. Transcription factors and transcriptional networks

A myriad of literature reports describe how environmental, developmental or hormonal signals
interfere with water relations in many plant species, and consistently act on regulation of individual
aquaporins. It is now required to go beyond these case-by-case studies and access a unified
understanding of the mechanisms involved and of their integration in whole plant responses.

With respect to aquaporin gene regulation, significant progress can be expected from genomic
studies. Several studies have addressed regulation of the whole aquaporin family (5, 34, 92, 116, 252,
340) and gene co-expression networks may reveal how regulation of individual aquaporins connects
with that of other isoforms or great physiological functions (4). Another important direction will be to
identify transcription factors that act as master regulators of aquaporin genes. A role for water stress
responsive factors belonging to the DREB (237) and ASR1 (240) families was recently established.
Regulation of aquaporin expression by additional ethylene (225) or abiotic stress responsive (325,
341) transcription factors is also emerging. In addition, plant aquaporins are predicted targets of
miRNAs in cotton (333) and potato (335). The impact of these and other epigenetic regulations on
plant water relations has not yet been evaluated.

Similar advances are foreseen at the protein level. Protein interaction network comprising
aquaporins (53, 123) should reveal as yet unknown functional interactions and help identify the
interplay of aquaporins with novel cellular functions.

## 3. General role of phosphorylation

Maintaining the plant water status under ever changing light, temperature, or water availability
requires constant cell and tissue hydraulic adjustments. Aquaporin phosphorylation provides a
crucial means for such rapid and reversible regulation, without the costs associated to protein
synthesis and degradation. Quantitative proteomics in *Arabidopsis* roots has revealed a general
correlation across abiotic and nutritional stimuli between Lpr and aquaporin phosphorylation (62).
This type of approach (200, 232, 234) will have to be refined for identifying the most relevant signals
and aquaporin isoforms involved in roots and other organs. Most importantly, the putative
regulations will have to be functionally validated by expression in transgenic plants of
phosphomimetic and phosphodeficient forms of the aquaporin isoform involved (232).

Exploring upstream signaling events also represent an important challenge for future studies.
A pioneering work recently identified SIRK1, a protein kinase involved in plant responses to sugar and
targeting PIPs (318). Hypothetical phosphorylation cascades triggered by ethylene and negatively
regulating aquaporin phosphorylation have recently been modelled (326). Yet, tremendous work
remains for identifying the numerous protein kinases and protein phosphatases that likely regulate
aquaporins. ABA-dependent phosphorylation may be of central importance (135), but we still ignore
the signaling components involved.
934 VII) PLANT GROWTH AND DEVELOPMENT

935 VII.A - Hydraulics of plant growth

936 1. Fundamental principles and role of aquaporins

937 Plants show continuous apical growth of their roots and shoots, through active cell multiplication in 938 meristems and subsequent cell expansion. These growth processes are remarkably plastic, providing 939 efficient means for long-term adaptation of plants to their environment. The plant water and 940 nutrient status in particular exert multiple controls on these processes.

941 Plant cell expansion has been formalized in the so-called Lockhart model. It is primarily driven 942 by the intracellular positive pressure (turgor) and mostly restricted by the cell wall that yields beyond 943 a turgor threshold (54). Since cell expansion requires a steady water intake, the model also predicts 944 that the rate of tissue growth can be restricted due to a drop in turgor, when water delivery, through 945 transcellular transport from the vasculature, encounters significant hydraulic resistances. These ideas 946 have been validated by measuring growth-associated water potential gradients (36). They provide a 947 frame for understanding the role of aquaporins in plant tissue growth. Accordingly, aquaporins were 948 shown to be strongly expressed in tissues that can be hydraulically-limiting during growth, in barley 949 or maize leaves or hypocotyls of *Arabidopsis* or castor oil plants (*Ricinus communis*) (68, 95, 166, 950 951 hydraulic conductivity (306).

952 A direct assessment of the role of aquaporins in tissue growth is experimentally challenging. It 953 was attempted using mercury, which reversibly inhibited root tip growth in maize and embryo 954 expansion in germinating broad bean seeds (110, 215). A hydraulic control of growth can also be 955 revealed at the whole plant level, when water supply from roots becomes limiting for leaf growth. A 956 role of aquaporins in this control was established by pharmacological inhibition of root water 957 transport using a panel of treatments (mercury, acid loads, H₂O₂) with distinct modes of action on 958 aquaporins. A good correlation between inhibition of Lpᵣ, drop of leaf cell turgor and leaf growth 959 retardation (67) was observed across these treatments. Interestingly, leaf growth was even more 960 sensitive than stomatal conductance to root hydraulics. Recently, this type of control was nicely 961 integrated in the context of diurnal variations of leaf growth (39). Growth was shown to be tightly 962 linked to circadian oscillations of plant hydraulic conductance and PIP transcript abundance in root. 963 In addition, these oscillations were amplified under drought, to adjust growth to water availability in 964 the soil and improve plant performance (39). These findings indicate that Lpᵣ regulation by numerous 965 environmental and hormonal stimuli provides a general means for quickly and reversibly adjusting 966 shoot growth to environmental constraints.

967 Whereas PIPs can play an obvious role in transcellular water transport towards expanding 968 tissues, vacuolar aquaporins (TIPs) also seem critical for plant growth. In *Arabidopsis* for instance, 969 expression of a specific TIP isoform (AtTIP1;1) was associated to cell expansion (166) and regulated 970 by the growth-promoting hormone gibberellic acid (GA₃) (228). Over-expression in the same plant of 971 a ginseng TIP orthologue enhanced overall plant growth through increased leaf cell size (154). It has 972 been suggested that TIPs critically contribute to osmoregulation and vacuolar differentiation in 973 expanding cells. Yet, the enhancement of tobacco protoplast division after TIP over-expression has 974 remained unexplained (219).
975

## 2. Particular developmental processes in shoots and roots

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Auxin was recently found to potently down-regulate the tissue-specific expression and function of most of root aquaporins. This regulation shed light onto a new role of aquaporins in root growth, to favour the emergence of secondary roots (226). In brief, physiological analyses in plants with various genetic alterations in PIP function were coupled to mathematical modelling to show that a fine spatial and temporal control of PIP expression favours water entry in the lateral root primordium. This flow is made at the expense of overlaying cells, thereby reducing their mechanical resistance and facilitating lateral root emergence. These results are crucial since they reveal for the first time a link between the capacity of the root to transport water and its ability to grow and potentially adjust its architecture to water availability.

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A role for aquaporins in local stimulation of growth has also been proposed in gravistimulated rice leaves. Accumulation of GA<sub>3</sub> on the abaxial side was shown to promote expression of a PIP gene (*OsRWC3*) thereby favoring local growth and leaf bending (108).

988

## 3. Growth of plants under water deficit

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1002

Water deficit usually results in leaf growth arrest, in part because down-regulation of root or leaf hydraulic conductance induce hydraulic limitations for growth (67, 223). Yet, there is a large array of responses to water deficit across all plant species. For instance, some cultivated plants such as maize or tomato exhibit an ABA-induced aquaporin up-regulation, to enhance soil water uptake and whole plant conductance and maintain plant growth under mild water deficit (39, 223). Under water limiting conditions, the so-called isohydric plants protect the leaf water status at midday through a tight stomatal regulation. By contrast, anisohydric plants have a less conservative strategy and favor gas exchange and photosynthesis at the expenses of water consumption. As long as water resources are not sharply limiting, the latter plants can exhibit better growth performance than the former. Transgenic tomato plants ectopically expressing a TIP gene or grapevine cultivars differing in drought tolerance (246, 298) have revealed that enhanced aquaporin activity typically confers an anisohydric behavior by favoring plant water transport and preventing stomatal closure. These phenomena certainly involve the general link found in many species, between root water transport (Lp<sub>r</sub>) and transpiration (142) (see VI.B-6).

1003

# VII.B - CO₂ fixation

1004

## 1. Mesophyll conductance to CO₂ (G<sub>m</sub>)

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1011

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Strikingly enough, the plant photosynthetic capacity, and therefore plant growth, are primarily limited by CO₂ delivery from the atmosphere to the sites of carboxylation in the chloroplasts (314). Whereas stomatal conductance (G<sub>s</sub>) accounts for gas diffusion through the stomatal pore, the mesophyll conductance to CO₂ (G<sub>m</sub>) corresponds to the subsequent transport of CO₂, from the sub-stomatal chamber to the chloroplast stroma (74, 314). G<sub>m</sub> can differ by more than ten-fold between species. It also varies according to physiological conditions such as water availability or atmospheric CO₂ (69). Several molecular and cellular entities such as cell walls, carbonic anhydrases or aquaporins have been proposed to contribute to G<sub>m</sub> (69). Each of these components may therefore underlie the genetic and physiological variations of G<sub>m</sub> in plants.

1014

## 2. Role of aquaporins

1015

1016

1017

Several lines of converging evidence indicate that aquaporins significantly contribute to G<sub>m</sub>. For instance, a 60-70% inhibition of G<sub>m</sub> by millimolar concentrations of mercuric chloride was reported in broad bean (*Vicia faba*) and French bean (*Phaseolus vulgaris*) leaves (283). Genetic studies in
transgenic tobacco (75, 293, 295), rice (100) or *Arabidopsis* (101) with enhanced or reduced PIP functions have revealed parallel variations in $G_m$. Finally, it was proposed that the dependency of $G_m$ to atmospheric CO₂ reflects a contribution of PIPs to $G_m$. High CO₂ would result in cell acidification, which in turn would reduce $G_m$ through pH-dependent gating of PIPs (73).

### 3. Open questions

Although a physiological role of PIPs in leaf CO₂ transport is emerging, this idea is not fully settled yet and requires a few notes of caution. Firstly, the methodology for measuring $G_m$ and its CO₂ dependency, in particular, is still disputed (73). Secondly, the $G_m$ variations observed after pharmacological or genetic manipulation of PIP functions are larger than those anticipated for the membrane path (69). Finally, some genetic studies are difficult to interpret because of confounding effects on stomatal gas exchanges ($G_s$) that would indirectly alter $G_m$. In these respects, ABA has recently been identified as a dual regulator of $G_s$ and $G_m$ (196) but its mode of action on $G_m$ is as yet unknown. Thus, studies where PIP genetic manipulation allows to uncouple effects on $G_m$ and $G_s$ are more reliable (261).

In any case, the role of aquaporins in CO₂ transport certainly represents a very exciting and significant field of research. Some biological membranes seem to have lower than expected permeability to CO₂, which would call for a significant contribution of membrane channels (125). More specifically, aerial plants have to achieve a critical tradeoff between water conservation and carbon fixation, and aquaporins might be at the center of these mechanisms. Future studies on this topic will surely shed light onto the molecular bases of $G_m$ and of its physiological regulations.

## VII.C - Nutrient allocation and toxicity

### 1. Boron (B)

B is necessary for plant growth. It is taken up from the soil and deposited in tissues to reinforce cell walls. *Arabidopsis* plants strongly react to B limiting conditions by decreased root length, burst in root hair development, and strong induction of the *AtNIP5*;1 gene (278) (FIGURE 12). Reverse genetic studies have shown that this aquaporin is central for root B uptake and plant growth under B limiting conditions. Maize *tsl1* plants mutated in a probable *AtNIP5*;1 orthologue also show growth defects in these conditions, with altered root growth and reduced size of the shoot apical meristem (65). In *Arabidopsis*, the *AtNIP6*;1 and *AtNIP7*;1 homologues also serve in B transport and mediate its allocation to shoots and pollen, respectively (148, 281). Additional aquaporins may be involved in B transport and, for instance, a role of XIPs in deposition of B within growing tissues has been proposed in tobacco (26).

Whereas B is an essential micronutrient, an excess in the soil can be toxic for the plant. Thus, the activity of some NIPs can become detrimental for plant growth. For instance, a quantitative trait locus for B toxicity in barley was associated to elevated expression of a NIP homologue in roots of the parental line showing the highest sensitivity to high B (257).

### 2. Silicon (Si)

Si, an abundant element of the soil, can accumulate in some plants (mostly cereals), to promote their growth and resistance to abiotic and biotic stresses. In particular, Si was shown to alleviate drought stress in sorghum, by enhancing root water uptake and aquaporin activity (161). Elegant genetic studies in rice have revealed a role for several NIP homologues (OsNIP2;1/Lsi1 and OsNIP2;2/Lsi6) in Si uptake and allocation to shoots (169, 323). As for B, Si transporting NIPs function as efflux channel.
**3. Other metalloids**

**VII.D - Plant reproduction**

**1. Flowers**
1103 reproductive development defects of the maize B transport mutant *tsl1* (65) indicate that, in addition
1104 to water, micronutrients such as B also are critical for proper growth and function of flowers.

1105 **2. Seeds**

1106 Seed maturation is accompanied by a profound reorganization of the vacuolar apparatus, with
1107 formation of numerous protein storage vacuoles. Although genetic evidence is lacking, the
1108 expression of seed-specific TIPs is thought to be crucial during this process (82, 276, 301). Studies in
1109 developing bean seeds also revealed a specific aquaporin equipment, for phloem-mediated import of
1110 water and nutrients into developing coat, and for water recycling in the xylem and its delivery to
1111 cotyledons (339).

1112 Seed germination is triggered by a rapid imbibition of desiccated tissues, and shows a second
1113 phase of water uptake associated to embryo growth. Mercury inhibition experiments have indicated
1114 that aquaporins may be limiting during the first phase of water uptake in species such as pea (305)
1115 whereas, in *Arabidopsis* or broad bean, they would contribute to embryo growth (215, 301). The
1116 transition from protein storage vacuoles to a large central vacuole, with a shift from TIP3 to TIP1
1117 expression, also seems crucial during this process.

1118 Despite the presumed importance of aquaporins in seeds, we note that, so far, genetic
1119 evidence is only available in rice. In this species, the knock-out and/or over-expression of OsPIP1;1
1120 and OsPIP1;3 was shown to alter both the rate and speed of germination (155, 158). Interestingly,
1121 the two genes are under the control of nitric oxide. The control of seed aquaporin function by
1122 osmotic or hormonal signals will deserve more efforts in future research.

1123 VIII) BIOTIC INTERACTIONS

1124 VIII.A - Rhizobium-legume symbiosis

1125 Besides plant responses to abiotic stimuli, aquaporins also serve in plant biotic interactions. The best
1126 described examples are root symbiosis.

1127 Nitrogen is, together with potassium and phosphorus, a major macronutrient of plants. It is
1128 most often limiting for plant growth, hence the intensive use of nitrogen fertilizers in agriculture.
1129 Legumes have acquired the remarkable capacity to develop symbiotic root nodules able to fix
1130 atmospheric nitrogen (N₂). For this, specific soil bacteria (*Rhizobiaceae*) infect plant root cells and
1131 differentiate into intracellular bacteroids, each surrounded by a plant symbiosome membrane (292).
1132 During root nodule differentiation in barrel clover (*Medicago truncatula*), a TIP1 homolog was shown
1133 to be transiently retargeted from the tonoplast to the symbiosome membrane, this process being
1134 necessary for functional nodule formation (84). The fully differentiated symbiosome membrane then
1135 expresses NIPs which transport both water and NH₃ (91, 112) and likely play a dual role in cell
1136 osmoregulation and nitrogen assimilation. One of these NIP homologs, named Nodulin-26 and
1137 present in soybean root nodule, was actually the first aquaporin identified in plants. Nodulin-26
1138 molecularly interacts with cytosolic glutamine synthase (184). Upon import of NH₄⁺/NH₃ into the
1139 plant cytosol by paths that still need to be unambiguously identified, this molecular interaction may
1140 optimize fixation of NH₄⁺ onto glutamate, to prevent a feedback leak of NH₄⁺/NH₃ to the
1141 peribacteroid space through Nodulin-26.
1142 VIII.B - Mycorrhizae

1143 Mycorrhizae represent a very common symbiotic interaction between soil fungi and plant roots.
1144 Depending on the two partners, the plant interface with the fungus is either intracellular (arbuscular
1145 mycorrhizae) or extracellular (ecto-mycorrhizae). In all cases, the mycelia optimize soil exploration
1146 and nutrient capture, whereas the plant root provides carbon metabolites for nutrient assimilation.
1147 Besides plant mineral nutrition, mycorrhizae seem to ameliorate plant water relations and stress
1148 responses. For instance, numerous reports indicate that, both in crops (maize, bean, soybean,
1149 lettuce) and trees (poplar), mycorrhizae can enhance plant tolerance to drought, flooding, cold or
1150 salinity stress (10, 17, 41, 179, 229). However, the molecular and physiological bases of these effects
1151 are not fully elucidated yet. For instance, root water transport and overall aquaporin expression were
1152 enhanced in poplar whereas an opposite response was seen in soybean and lettuce (179, 229). In the
1153 latter two species, arbuscular mycorrhizae may help the plant anticipate root responses to stress. In
1154 maize, interactive effects of mycorrhizae with plant ABA enhance root aquaporin inhibition and
1155 water conservation under drought (243).

1156 The role of plant aquaporin-mediated solute transport in arbuscular mycorrhizae was
1157 recently examined (17). Gene profiling indicated that, in symbiotic roots, glycerol export from the
1158 plant to the microbe, and NH4+/NH3 import into the plant, for storage and detoxification, may be
1159 enhanced through activation of proper plant aquaporin genes. By contrast, the transport of B and Si
1160 would be down-regulated. Indeed, the B released by the fungus can be toxic for the plant, and Si is
1161 known to antagonize mycorrhizal infection.

1162 A current challenge is now to characterize the aquaporin equipment of fungi, a task that was
1163 accelerated through fungal genome sequencing (63, 149, 322). Similar to their plant counterparts,
1164 aquaporins of arbuscular or ecto-mycorrhizal fungi such as *Glomus intraradices* or *Laccaria bicolor*,
1165 respectively, appear to play a crucial role in both water and nutrient transport. An ultimate challenge
1166 will be to understand the molecular dialog between plant and fungal cells, and how this coordinates
1167 expression of aquaporins and complementary transport systems between the two partners (9). Even
1168 though the underlying mechanism are not known, it was recently shown that genetic manipulation of
1169 aquaporin expression in *Laccaria bicolor* alters expression of the plant host aquaporins (322).

1170 VIII.C - New directions: investigating beneficial or pathogenic interactions

1171 Recent studies on plant root symbiosis have revealed how aquaporins can contribute to a functional
1172 dialog between transport proteins of distinct organisms. Yet, many other biotic interactions remain
1173 to be explored to ultimately apprehend the full ecological integration of aquaporin functions.

1174 For instance, the plant-growth-promoting rhizobacterium *Bacillus megatorium* exerts
1175 ameliorative effects on plant salt tolerance. Accordingly, it enhances Lpr in maize and modifies
1176 aquaporin expression in both control and salt stress conditions (183). Yet, the precise modes of
1177 aquaporin regulation and the role of bacterial signals, possibly auxin, remain unknown. The plant
1178 hormone methyl jasmonate also represents an important signal in plant defense. It was shown to
1179 enhance aquaporin activity in roots of French bean, tomato and *Arabidopsis* through interaction with
1180 calcium- and/or ABA-dependent signaling pathways (253).

1181 The effects of pathogenic infection on aquaporin expression and their significance in plant
1182 disease will also deserve a specific attention. One of most striking example is the induction of
1183 *Arabidopsis* AtTIP1;1 expression in giant root cells induced upon nematode infection (220). This
1184 induction may favor the osmoregulation of these cells that serve as feeding sites for the pathogen.
1185 Infection of soybean leaves by *Pseudomonas syringae* resulted after 8h in down-regulation of 24 out

1186 32 aquaporin genes (342) but the significance of this regulation was not assessed.

1187 **IX) AQUAPORINS AND PLANT GENETIC IMPROVEMENT**

1188 **IX.A - Success stories and failures**

1189 Water relations and mineral nutrition represent major traits in crop improvement. Aquaporins, which
1190 provide genetic and molecular tools to act on these traits, are therefore attractive targets for plant
1191 molecular breeders (180). Genetic manipulation of PIPs, TIPs and NIPs has now been explored in
1192 many plant species, with varying rates of success.

1193 Transgenic strategies aimed at altering aquaporin function have first been developed in
1194 herbaceous species, leading to contrasting effects on plant growth and stress response. Ectopic
1195 expression of an aquaporin in a heterologous plant species seems hazardous, since the fine
1196 regulations that govern each individual isoform in the native plant may not work in a distinct
1197 transgenic species. Yet, overexpression of a *Vicia faba* PIP1 (*VfPIP1*) or a wheat PIP2 (*TaAQP7*) in
1198 *Arabidopsis* and tobacco, respectively, enhanced plant drought resistance (55, 338). Identification of
1199 stress-regulated aquaporin isoforms and subsequent genetic manipulation in the same species seems
1200 to be a more solid approach. For instance, constitutive expression of stress-responsive *S/TIP2;2*
1201 enhanced the growth performance of transgenic tomato plants under both normal and water stress
1202 conditions by favoring their anisohydric behavior (246). OsPIP1;3 (RWC3) was initially identified as
1203 stress-induced in a upland rice cultivar. Its expression in an lowland rice cultivar, using an engineered
1204 stress-induced promoter, conferred on transgenic plants drought avoidance properties (such as
1205 maintenance of leaf water potential) reminiscent of those observed in the upland cultivar (153). In
1206 rice again, tolerance of root and leaf growth to salt stress and ameliorated germinative properties of
1207 seeds were obtained upon constitutive expression of OsPIP1;1, provided that the transgene was
1208 expressed at a moderate level (155).

1209 In addition to these few success stories, the literature abounds with more mitigated reports.
1210 These works are informative with respect to aquaporin physiology but represent clear failures in
1211 terms of biotechnology. For instance, tobacco plants overexpressing *Arabidopsis* AtPIP1;2 grew
1212 better than control plants in normal conditions but became dramatically more sensitive to water
1213 deprivation (3), probably because of a stomatal deregulation. Expression of cucumber and figleaf
1214 gourd aquaporins in *Arabidopsis* had either beneficial or detrimental effects on plant survival and
1215 seed germination, whether plant where subjected to a dehydration (mimicked by PEG application) or
1216 a salt (NaCl) stress (117).

1217 By comparison to herbaceous crops, the genetic improvement of woody plants poses special
difficulties due to slower growth and longer reproduction cycles. Yet, with the emergence of genomic
tools and reverse genetics, aquaporin functions and the potentialities of their genetic alteration are
1219 now being explored in these species. For instance, overexpression of a root PIP (VvPIP2;4N) in
1220 transgenic grapevine (227) enhanced root hydraulics, transpiration and shoot growth, and therefore
1221 water consumption under control conditions whereas the plant exhibited a normal water
1222 conservation response under water stress. In poplar, RNAi was recently used as an efficient means
1223 for down-regulating expression of several PIP1 homologs. Whereas no major phenotype was
1224 observed in transgenic trees with fast growth under no stress conditions, trees under water stress
1225 unfortunately showed severe defects in recovery from embolism (259, 261). *Eucalyptus* is another
1226
1227 forest tree of great use in the paper industry. Aquaporin function was explored in the species using
1228 transgenesis (288). Finally, targeting ethylene-regulated aquaporins in *Hevea* may help improve latex
1229 production (289).

1230 IX.B - Mechanisms

1231 The phenotype of plants with genetically altered aquaporins is usually complex to decipher, as it
1232 integrates far more than direct effects of the manipulated aquaporin on tissue hydraulics or carbon
1233 fixation. For instance, several studies have revealed that ectopic expression of a foreign aquaporin
1234 gene can perturb the expression pattern of endogenous aquaporin genes, thereby resulting in
1235 unpredictable beneficial or detrimental effects on plant growth and stress responses (117, 288).
1236 Heteromerization of an artificially expressed aquaporin with endogenous ones may also result in
1237 dominant negative effects on plant water transport (270). In addition, altered water relations can
1238 influence other physiological responses to stress, which in turn contribute to most of plant
1239 phenotype. For instance, PIP overexpression in grapevine may have disturbed water stress sensing in
1240 root, thereby enhancing ABA synthesis, specifically under water stress (227). These hormonal effects
1241 likely explain why water conservation was improved in these conditions (227). At high
1242 concentrations, Na⁺ is a toxic ion for plants. Controlling its accumulation and compartmentalization is
1243 therefore critical in plants under salt stress. In *Arabidopsis*, over-expression of a wheat NIP
1244 homologue (81) or a TIP from the halophyte *Thelungiella salsuginea* (312), reduced salt loading or
1245 favored its vacuolar accumulation, respectively. In both cases, plant stress tolerance was improved.
1246 ROS metabolism, which is central to plant stress responses, can also be altered after aquaporin
1247 genetic manipulation. Overexpression of a wheat PIP2 homologue (*TaAQP7*) in tobacco resulted in
1248 enhanced superoxide dismutase and catalase activities through an unknown mechanism (338),
1249 thereby contributing to drought tolerance. Conversely, an *Arabidopsis* line inactivated for *TIP1;1* and
1250 *TIP1;2* showed a reduced catalase activity and higher anthocyanin content that somewhat mimicked
1251 a response to high light. This phenotype was tentatively associated to the capacity of the two TIP
1252 homologues to channel H₂O₂ (258). In most studies, information on root and shoot hydraulics, but
1253 also leaf morphology, stomatal density, and shoot/root ratio is lacking to fully interpret how
1254 aquaporin manipulation can interfere with plant growth properties. Yet, elegant grafting experiments
1255 using wild-type and transgenic tobacco showed that overexpression of *NtAQP1* in roots was
1256 sufficient to sustain a high transpiration in wild-type shoots. In contrast, the beneficial effects of
1257 *NtAQP1* on stomatal conductance and photosynthesis required its expression in shoots (245).

1258 IX.C - Targeting micronutrients

1259 Besides water relations and growth under drought or salt stress, the plant micronutrient status
1260 represents another, easier target for aquaporin genetic manipulations, which, however has barely
1261 been explored. Thus, enhanced expression of AtNIP5;1 and AtTIP5;1 in *Arabidopsis* improved plant
1262 tolerance to low B and high B, respectively (129, 222). In the latter case, the effects were likely due
1263 facilitated storage of B in the vacuole. Reduced expression of NIPs that transport B at the plasma
1264 membrane should also enhance plant tolerance to high B toxicity (257). We note however that, as for
1265 water transport, ectopic expression of aquaporins transporting B and Si can lead to detrimental
1266 effects on growth. Thus, organ- or tissue-specific expression seems to be crucial, to prevent mis-
1267 localization of these micronutrients (129, 201).
1268 **X) CONCLUSIONS AND PERSPECTIVES**

1269 **X.A - Conclusions**

1270 Two decades after their discovery, it is now established that aquaporins play a broad role in plant physiology. Genetic and physiological studies, in support of gene expression data and functional characterization in heterologous systems, have revealed that, despite their multiplicity, plant aquaporins can individually fulfill multiple functions. For instance, some PIP1s seem to play a dual role in water and CO₂ transport in *Arabidopsis* and tobacco (75, 230, 264, 267). In the former species, a single PIP2 isoform (AtPIP2;1) is involved in root water uptake, lateral root formation and water transport in leaf veins (226, 232). Therefore, a next challenge will be to dissect, at least for a few representative isoforms, the cell-specificity of aquaporin functions and regulations. One direction would be targeted proteomics, to access cell-specific aquaporin phosphorylation profiles and interacting partners. Transgenic plants expressing cell-type specific reporters (232, 264) and aquaporin knock-out plants with cell-specific complementation (232, 244) will be crucial in these studies. Beyond these generic approaches, we believe that a few, more specific topics will deserve a specific attention.

1283 **X.B - Emerging research directions**

1284 **1. Aquaporins and plant cell signaling: interplay with ROS and hormones**

1285 Through their multiple transport functions and regulations, aquaporins emerge as critical nodes that integrate cell metabolism and signaling into whole plant responses to environmental and hormonal signals. The interplay of aquaporins with ROS appears as a particularly central topic. On the one hand, ROS mediate the effects of salt, salicylic acid, and cold (33, 145) on water transport and aquaporin regulation in roots. Yet, we do not understand why ROS have contrasting effects in bean (21) and *Arabidopsis* roots (35). On the other hand, the capacity of plant aquaporins to transport H₂O₂ points to emerging roles in cell signaling and ROS detoxification. The former has been established in animals (192) and the latter is supported by recent studies in transgenic plants. The regulation of aquaporins by hormones in plants under environmental stress will also deserve more attention. For instance, ethylene may play crucial role for water transport regulation during anoxia (126) or potassium starvation. Finally, the circadian regulation of aquaporins and the significance of hydraulics as an input or output signal of the clock will represent an exciting challenge in coming years (39, 279). Mutants such as *early flowering 3* will allow investigating how aquaporin function is coupled to the circadian clock (279). In addition, understanding the synchronizing role of light but also ABA on oscillating aquaporin functions may provide clues on how plant growth is optimized according to soil water ability and transpiration demand (39, 279).

1301 **2. Role of intracellular aquaporins**

1302 Whereas it definitely pertains to original plant specific functions, the role of intracellular aquaporins, has remained a kind of black box. Since reverse genetics of TIPs and SIPs has failed to reveal macroscopic phenotypes (19, 174, 258), cellular phenotypes will have to be inspected in closer details. In particular, an accurate monitoring of compartmental volumes in osmotically-challenged cells will be required to establish the putative role of these aquaporins in cell osmoregulation. Also, the actual substrates of these aquaporins are as yet undetermined. It was suggested that SIPs may transport ethylene across the ER membrane (174) but, to our knowledge, this hypothesis has not
1309 been further explored. Finally, the role of PIPs in CO₂ transport across the chloroplast envelope is
1310 indicated by a single study in tobacco (295) and definitely deserves more attention.

1311 **3. Exploring more diverse plant systems**

1312 While initially performed in a few plant model species (*Arabidopsis*, tobacco) or crops (maize, rice),
1313 studies on aquaporins are now expanding to more and more plant species. Plant biodiversity is
1314 definitely worth being further explored. In particular, extremophile plants such as resurrection plants
1315 or halophytes (177, 300, 312) may reveal novel physiological regulations. In ice plant, aquaporin
1316 function is dramatically changed during the salt-induced switch from C3 to CAM metabolism (134,
1317 302). Original information is also expected from unicellular photosynthetic organisms such as
1318 *Synecocystis* (12) or *Chlamydomonas reinhardtii* (138), from mosses (154) or representative species
1319 of early evolved plants (8). In particular, a recent studies in horsetail (*Equisetum arvense*) a primitive
1320 vascular plant that was identified for its remarkably high Si content allowed identification of a new
1321 subfamily of aquaporin Si channels (89).

1322

1323 By delineating ever expanding fields in plant integrative biology, aquaporins have been fascinating
1324 research objects over the last two decades. This is quite not finished: we have outlined here a few
1325 research directions which, we believe, are particularly relevant and have been somewhat neglected.
1326 We are confident that these and other directions will provide exciting discoveries and establish
1327 further the crucial role of aquaporins in plants.

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1901 214. Noronha H, Agasse A, Martins AP, Berny MC, Gomes D, Zarrouk O, Thiebaud P, Delrot S, Soveral G, Chaumont F, and Geros H. The grape aquaporin VvSIP1 transports water across the ER membrane. *J Exp Bot* 65: 981-993, 2014.

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1915 219. Okubo-Kurihara E, Sano T, Higaki T, Kutsuna N, and Hasezawa S. Acceleration of vacuolar regeneration and cell growth by overexpression of an aquaporin NtTIP1;1 in tobacco BY-2 cells. *Plant Cell Physiol* 50: 151-160, 2009.

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FIGURE LEGENDS

**FIGURE 1.** Short- and long-distance transport of water in a vascular plant. During transpiration, an intense stream of water occurs from the roots to the shoots. Soil water flows radially across concentric cell layers in the root before reaching the xylem vessels and being exported to the shoots. In leaves, water flows out of vascular tissues and reaches substomatal chambers where it diffuses as a gas (vapor) through the stomata.

**FIGURE 2.** Phylogenetic tree of the *Arabidopsis* and rice aquaporin families. The evolutionary history of aquaporins of the two plant species was analyzed using a MEGA4 software (280). It was first inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 189 positions in the final dataset.

**FIGURE 3.** Molecular mechanisms of PIP gating. The structure of spinach SoPIP2;1 was solved in an open (A: PDB code 2B5F) and a closed (B: PDB code: 1Z98) conformation (286). In the open conformation (A), the His193 (green) is not protonated and loop *D* is distal from the other cytoplasmic loop (loop *B*). In the closed conformation (B), the protonation of His193 allows interaction with an acidic residue (Asp28, in yellow) of the N-terminus. This in turns drives a conformational change of loop *D* and occlusion of the pore by displacement, into the cytosolic pore mouth, of the hydrophobic side chain of Leu197 (in pink). Binding of divalent cations (Cd²⁺ in the atomic structure, in yellow) also involves Asp28 and an adjacent acidic residue (Glu31, in yellow). Loop *D* is thereby stabilized in the closed pore conformation through a network of H-bond and hydrostatic interactions, involving Arg118 (yellow). In this model, phosphorylation of loop *B*, at Ser115 (in purple) would disrupt this network of interactions and unlock loop *D* to allow the open conformation.

**FIGURE 4.** Multiple covalent post-translational mechanisms acting on plant plasma membrane aquaporins. The figure shows a schematic representation of *Arabidopsis* AtPIP2;1 with its 6 transmembrane domains (1-6), five connecting loops (A-E), and N- and C-terminal tails bathing in the cytosol. The indicated modifications were mostly determined by mass spectrometry. They include: cleavage of the initiating methionine (cross), di-methylation (Met Met) of a Lys residue (K3), mono-methylation (Met) of a Glu residue (E6) (254), deamidation (Dea) of Gln and Asn residues (Q14, Q19, N286) (62) and ubiquitination (Ub), the ubiquitinated residue(s) being unknown (143). Phosphorylation of some Ser residues (S280 and S283) was established experimentally (62, 234). By contrast, the phosphorylation of loop *B* at S121 was inferred from studies on spinach SoPIP2;1 (122, 286). Some enzymes involved in covalent modification of PIPs have been identified. They include: methyl-transferases SDG7 (At2g44150) and OMTF3 (At3g61990) (247), a U3 ligase (143) and several protein kinases including Mg²⁺- and Ca²⁺-dependent protein kinases (268) and SIRK1 (At5g10020) (318). The figure is color-coded to show the enzymatic targets and the main molecular and cellular
function associated to the post-translational modification. Thus, post-translational modifications
interfere with the subcellular localization (red), gating (green) and degradation of the aquaporin
(orange). The functional role of PIP deamidation is unknown.
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**FIGURE 5.** Localization of aquaporins in the plant plasma and vacuolar membranes. Transgenic *Arabidopsis* plants expressing AtPIP2;1 fused to the green fluorescent protein (PIP2;1-GFP) (A), or co-expressing PIP2;1-GFP and AtTIP1;1 fused to the fluorescent protein mCherry (TIP1;1-mCherry) (B), were observed by laser scanning confocal microscopy. A: the figure shows the plasma membrane localization of PIP2;1-GFP in a cross section of a root (left panel) with indicated cell layers (ep: epidermis; co: cortex; en: endodermis; st: stele), and in epidermal cells of cotyledons (right panel). B: the figure shows the localization in root epidermal cells of PIP2;1-GFP and TIP1;1-mCherry, in the plasma membrane and vacuolar membrane (tonoplast), respectively. The arrows indicate cytoplasmic strands formed within grooves at the surface of the invaginated vacuole, and the asterisk indicates a nucleus skirted by the tonoplast. Bar size = 20 µm.
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**FIGURE 6.** Subcellular dynamics of PIPs and TIPs. A: Targeting of aquaporins to the plasma membrane and tonoplast. Both PIPs and TIPs follow the secretory system to be targeted to their final destinations. All isoforms are synthesized in the endoplasmic reticulum (ER). Specific signals or mechanisms are requested for the exit of PIPs out of this compartment. These include diacidic (D/EXD/E) or LXXXA motifs, or phosphorylation (Phosp) for PIP2s, or physical interactions with PIP2s (PIP1s/PIP2s) for PIP1s. The existence of similar mechanisms or motifs is not documented for TIPs. The targeting pathways followed by TIP1s and TIP2/3s are sensitive to brefeldin A (BFA) or C834, respectively. The Golgi apparatus (GA) and Trans Golgi Network (TGN) continuum provide intermediate compartments for trafficking of PIPs, prior to their exocytosis towards the PM. In contrast, TIP1s traffic through the GA/TGN and multivesicular body (MVB) compartments before reaching the tonoplast. Note that, in plants, MVB is also termed prevacuolar compartment (PVC) or late endosomal compartment (LE). The precise targeting path followed by TIP2/3s is not yet known. Once at the plasma membrane, PIPs undergo constitutive cycling, through endocytosis from the cell surface towards early endosomes (EE) using a clathrin-dependent pathway and subsequent exocytosis back to the PM. The TGN and the EE are considered to form a single compartment. The syntaxins (SYP) that are known to play a role in some steps of PIP or TIP trafficking are indicated. B: Subcellular dynamics of PIPs, with mobilization from the plasma membrane to intracellular compartments, in salt-stressed root cells. The effects of salt are mediated through ROS which enhance the lateral mobility of PIPs at the cell surface and their partitioning in membrane micro-domains. PIP cycling, by endocytosis through clathrin-dependent and clathrin-independent pathways and exocytosis, is also enhanced by ROS. Some of the internalized PIPs are directed toward the MVB and spherical bodies. Same abbreviations as in A. C: Osmotic stress-induced degradation of PIPs. In addition to enhanced cycling, PIPs undergo ubiquitination or interaction with multi stress regulator proteins. As a consequence, PIPs are directed toward the proteasome and vacuolar degradation pathways. The latter pathway involves autophagosomes (see main text for details). Same abbreviations as in A.
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**FIGURE 7.** Large-scale determination of plant aquaporin interactomes. The figure refers to two independent studies using split-Ubiquitin (Split-Ub) (123) and yeast two-hybrid (Y2H) (53), respectively, that were used for genome-wide identification of plant aquaporin-interacting proteins
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