2344 in *Arabidopsis*. A: number of aquaporin-interacting partners. In the Split-Ub screen, an interaction
2345 between two partners was validated provided that the interaction was reproducible in both a
2346 primary and a secondary interaction screen. Thus, 22 aquaporin isoforms showed interactions with at
2347 least one protein. In contrast, 10 isoforms (PIP1;1, PIP1;5, PIP2;6, TIP1;2, TIP2;1, TIP3;1, NIP1;2,
2348 NIP3;1, NIP5;1, SIP1;1) did not exhibit any interacting proteins. No information is available for PIP2;4,
2349 TIP3;2 and NIP4;2. For more details, see https://associomics.org. The Y2H screen identified
2350 interacting proteins for 18 aquaporins. For more details see (53). B, C, D: Gene Ontology (GO)
2351 classification of proteins interacting with aquaporins using Panther (http://www.pantherdb.org/).
2352 Bold characters indicate major GO terms.
2353

2354 FIGURE 8. Putative functions of aquaporins in the chloroplast. Several PIPs and TIPs have been
tentatively localized in the inner envelope and thylakoid membranes. They may contribute to water
2355 transport into the stroma and thylakoid lumen. In the latter compartment, photosynthesis leads to
2356 oxidation of water to molecular oxygen. Changes in incident light can result in adjustments of
2357 thylakoid and overall chloroplast volumes. A role of PIP1s in CO₂ transport across the inner envelope
2358 membrane is also indicated. Carbon fixation occurs in the stroma through carboxylation reactions
2359 within the Calvin-Benson cycle. Finally, ROS can be formed as by-products of photosynthetic
2360 activities. A putative role of PIP and TIP in facilitating H₂O₂ export from the chloroplast is shown.
2361
2362

2363 FIGURE 9. Water transport and aquaporin expression in roots. A: Schematic representation of an
2364 *Arabidopsis* root cross section (red: epidermis; blue: cortex; green: endodermis; inner red: pericycle
2365 tissue; brown: xylem vessels). Water flows through the cell-to-cell and apoplastic paths are
2366 represented by blue arrows. B-D: radial and longitudinal cuts of *Arabidopsis* roots expressing
2367 *AtPIP1;2-GUS* (*B*; (230)), *AtPIP2;2-GUS* (*C*; (119)) or *AtPIP2;1-GUS* (*D*; (226)) transgenes, which allow
2368 expression of β-glucuronidase (*GUS*) reporter gene under the control of the indicated PIP promoters.
2369 These pictures show how various aquaporin isoforms exhibit different expression patterns although
2370 all are expressed in the stele. Bars = 50µm (*B*), 50µm (*C*) or 25µm (*D*).

2371
2372 FIGURE 10. Aquaporin-dependent downregulation of root water transport during abiotic stresses.
2373 The figure redrawn from the work in (42) shows sap flow (Jv) exuded by detached root systems of
2374 wheat. Roots of control plants treated first with 50 µM HgCl₂ and then with 5 mM DTT highlight the
2375 role of the aquaporin-dependent path in root water transport (blue filled circles). Jv in roots of
2376 nitrogen (N)- and phosphorus (P)-deprived plants (red and green open circles, respectively) is much
2377 lower and shows no sensitivity to HgCl₂inhibition. The conclusion of this landmark experiment is that
2378 inhibition of root water transport in plants under N- and P- deficiency is mediated through aquaporin
2379 regulation.
2380

2381 FIGURE 11. Water transport and aquaporin expression in leaves. A: Schematic representation of an
2382 *Arabidopsis* leaf section (red: epidermis; blue: mesophyll; green: parenchyma; brown: bundle sheath;
2383 light green and purple: conductive tissues). Water flows through the cell-to-cell and apoplastic paths
2384 are represented by purple arrows. B: staining of whole organ or sections in *Arabidopsis* leaves
2385 expressing the *PIP1;2-GUS*, *PIP2;1-GUS*, *PIP2;6-GUS* or *pPIP2;7-GUS* transgenes (230, 232). The
2386 pictures show a unique leaf expression pattern for each isoform. Black bars = 2.5mm; red bars =
2387 0.1mm.

2388
2389

**FIGURE 12.** Key experiments showing the role of AtNIP5;1 in B transport (278). A: Time-dependent

2390

accumulation of *AtNIP5;1* mRNA in roots of B-deprived plants, as revealed by quantitative RT-PCR. B: Swelling assay in *Xenopus* oocytes incubated in an isotonic B solution, showing that, by contrast to

2391

*AtPIP2;1*, *AtNIP5;1* is permeable to B. C: The reproductive growth of a *nip5;1* knock-out mutant

2392

(*nip5;1-2*) is specifically altered under B-limiting conditions. For more experimental details see the

2393

original publication (278).

2394

2395

**FIGURE 13.** Cell-specific and subcellular localization of pollen TIPs. Expression in transgenic

2396

*Arabidopsis* of fluorescent protein fusion constructs show that *AtTIP5;1* (A) and *AtTIP1;3* (D) are

2397

specifically expressed in the sperm and the vegetative cell of pollen, respectively. Co-localization (C) of TIP5;1-mCherry (A) with an *Arabidopsis* vacuolar H⁺-ATPase subunit E construct (VHA-E1-GFP) (B)

2398

2399

indicates the vacuolar localization of *AtTIP5;1*. In a germinating pollen grain (see transmission image

2400

in *E*), *TIP1;3-GFP* shows an intricate labelling of tonoplast and endoplasmic reticulum membranes.

2401

The position of two vacuoles is indicated (v). Bar size = 10 µm.

2402

2403

2404

2405
<table><caption>Table 1. Diversity of aquaporin gene family in plants.</caption><thead><tr><th>Species</th><th>Common name</th><th>PIPs</th><th>TIPs</th><th>NIPs</th><th>SIPs</th><th>XIPs</th><th>HIPs</th><th>GIPs</th><th>Total</th><th>Ref.</th></tr></thead><tbody><tr><td>Selaginella moellendorffii</td><td>Spike moss</td><td>3</td><td>2</td><td>8</td><td>1</td><td>3</td><td>2</td><td></td><td>19</td><td>(8)</td></tr><tr><td>Physcomitrella patens</td><td>Moss</td><td>8</td><td>4</td><td>5</td><td>2</td><td>2</td><td>1</td><td>1</td><td>23</td><td>(59)</td></tr><tr><td>Oryza sativa</td><td>Rice</td><td>11</td><td>10</td><td>10</td><td>2</td><td></td><td></td><td></td><td>33</td><td>(252)</td></tr><tr><td>Arabidopsis thaliana</td><td>Mouse ear-cress</td><td>13</td><td>10</td><td>9</td><td>3</td><td></td><td></td><td></td><td>35</td><td>(121, 235)</td></tr><tr><td>Solanum lycopersicum</td><td>Garden tomato</td><td>14</td><td>11</td><td>12</td><td>4</td><td>6</td><td></td><td></td><td>47</td><td>(239)</td></tr><tr><td>Populus trichocarpa</td><td>Black Cottonweed</td><td>15</td><td>17</td><td>11</td><td>6</td><td>6</td><td></td><td></td><td>55</td><td>(93)</td></tr><tr><td>Glycine max</td><td>Soybean</td><td>22</td><td>23</td><td>13</td><td>6</td><td>2</td><td></td><td></td><td>66</td><td>(334)</td></tr><tr><td>Gossypium hirsutum</td><td>Upland cotton</td><td>28</td><td>23</td><td>12</td><td>7</td><td>1</td><td></td><td></td><td>71</td><td>(224)</td></tr></tbody></table>

The table shows for all plant species, the genome of which was fully sequenced, the number of homologues

present in each of the indicated aquaporin subclasses.

2410

2411

2412
<table><caption>Table 2. Functional expression and substrate specificity of representative plant aquaporins</caption><thead><tr><th>Subclass</th><th>Isoform</th><th>Substrate</th><th>Expression system</th><th>Transport assay</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">PIP</td><td>AtPIP2;1</td><td>water</td><td>Proteoliposome</td><td>Shrinkage</td><td>(304)</td></tr><tr><td>AtPIP2;1</td><td>H<sub>2</sub>O<sub>2</sub></td><td>Yeast</td><td>Toxicity growth assay</td><td>(66)</td></tr><tr><td>AtPIP2;2</td><td>water</td><td>Xenopus oocyte</td><td>Swelling</td><td>(287)</td></tr><tr><td>NtAQP1</td><td>glycerol</td><td>Xenopus oocyte</td><td>Radiolabelling</td><td>(25)</td></tr><tr><td>NtAQP1</td><td>CO<sub>2</sub></td><td>Xenopus oocyte</td><td>Intracellular pH</td><td>(293)</td></tr><tr><td>NtAQP1</td><td>CO<sub>2</sub></td><td>Yeast</td><td>Intracellular pH</td><td>(221)</td></tr><tr><td>NtAQP1</td><td>CO<sub>2</sub></td><td>Planar lipid bilayer</td><td>Local pH</td><td>(294)</td></tr><tr><td>AtTIP1;1</td><td>water</td><td>Xenopus oocyte</td><td>Swelling</td><td>(187)</td></tr><tr><td>NtTIPa</td><td>urea</td><td>Xenopus oocyte</td><td>Radiolabelling</td><td>(86)</td></tr><tr><td>NtTIPa</td><td>glycerol</td><td>Xenopus oocyte</td><td>Radiolabelling</td><td>(86)</td></tr><tr><td rowspan="4">TIP</td><td>AtTIP1;2</td><td>H<sub>2</sub>O<sub>2</sub></td><td>Yeast</td><td>Intracellular fluorescence</td><td>(29)</td></tr><tr><td>TaTIP2</td><td>NH<sub>3</sub></td><td>Yeast</td><td>Extracellular pH</td><td>(115)</td></tr><tr><td>ZmTIP1;1</td><td>H<sub>2</sub>O<sub>2</sub></td><td>Yeast</td><td>Toxicity growth assay</td><td>(17)</td></tr><tr><td>AtTIP2.3</td><td>NH<sub>3</sub></td><td>Xenopus oocyte</td><td>Radiolabelling</td><td>(164)</td></tr><tr><td rowspan="7">NIP</td><td>AtNIP5;1</td><td>B(OH)<sub>3</sub></td><td>Xenopus oocyte</td><td>Intracellular dosage</td><td>(278)</td></tr><tr><td>OsNIP2;1</td><td>Si(OH)<sub>4</sub></td><td>Xenopus oocyte</td><td><sup>68</sup>Ge-radiolabelling</td><td>(169)</td></tr><tr><td>AtNIP5;1</td><td>As(OH)<sub>3</sub></td><td>Xenopus oocyte</td><td>Intracellular dosage</td><td>(193)</td></tr><tr><td>ZmNIP2;1</td><td>GeO<sub>2</sub></td><td>Yeast</td><td>Toxicity growth assay</td><td>(17)</td></tr><tr><td>BjNOD26</td><td>water</td><td>Proteoliposome</td><td>Shrinkage</td><td>(112)</td></tr><tr><td>BjNOD26</td><td>NH<sub>3</sub></td><td>Proteoliposome</td><td>Internal pH</td><td>(112)</td></tr><tr><td>VvSIP1</td><td>water</td><td>Yeast</td><td>Shrinkage</td><td>(214)</td></tr><tr><td>VvSIP1</td><td>water</td><td>Proteoliposome</td><td>Shrinkage</td><td>(214)</td></tr><tr><td rowspan="2">XIP</td><td>NtXIP1;1</td><td>H<sub>2</sub>O<sub>2</sub></td><td>Yeast</td><td>Toxicity growth assay</td><td>(26)</td></tr><tr><td>PtXIP2;1</td><td>water</td><td>Xenopus oocyte</td><td>Swelling</td><td>(162)</td></tr></tbody></table>

This table is not intended to be exhaustive. It illustrates using selected examples the diversity of expression systems and transport assays used to determine the wide range of substrates found for members of each plant aquaporin subclass.
2414

2415

<table><caption>Table 3. Molecular and functional characterization of aquaporin-interacting proteins</caption><thead><tr><th rowspan="2">Aquaporin</th><th colspan="2">Interacting protein</th><th rowspan="2">Method</th><th rowspan="2">Reference</th></tr><tr><th>Name</th><th>Function</th></tr></thead><tbody><tr><td>ZmPIP1;1,<br>ZmPIP1;2, ZmPIP1;6</td><td>ZmPIP2;1 and/or<br>ZmPIP2;5</td><td>aquaporin</td><td>FRET/FLIM</td><td>(331)</td></tr><tr><td>RhPIP1;1</td><td>RhPIP2;1</td><td>aquaporin</td><td>Split-Ub, BiFC</td><td>(47)</td></tr><tr><td>GhPIP2;6</td><td>GhPIP1</td><td>aquaporin</td><td>Y2H, BiFC</td><td>(147)</td></tr><tr><td>AtPIP2;1</td><td>Rma1H1</td><td>E3 Ub ligase</td><td>Y2H<br>Pull-down</td><td>(143)</td></tr><tr><td>ZmPIP2;5</td><td>ZmSYP121</td><td>syntaxин</td><td>Affinity purification,<br>BiFC</td><td>(24)</td></tr><tr><td>AtPIP2;7</td><td>AtSYP61 (At1g28490)</td><td>syntaxин</td><td>Pull-down, BiFC,<br>Split-Ub</td><td>(96)</td></tr><tr><td>AtPIP2;7</td><td>AtSYP121 (At3g11820)</td><td>syntaxин</td><td>Pull-down, BiFC,<br>Split-Ub</td><td>(96)</td></tr><tr><td>AtPIP2;7</td><td>TSPO (At2g47770)</td><td>Tryptophan-rich<br>sensory protein/<br>translocator</td><td>Pull-down, BiFC</td><td>(98)</td></tr><tr><td>AtPIP1;3, 2;2, 2;6,<br>2;1, 1;1, 1;4, 1;5,<br>2;7, 2;4</td><td>SIRK1 (At5g10020)</td><td>LRR-receptor-like<br>kinase family,</td><td>Pull-down</td><td>(318)</td></tr><tr><td>NOD26</td><td>Soybean GS1 β1, β2 γ1,<br>γ2</td><td>Glutamine synthase</td><td>Split-Ub, BiFC</td><td>(184)</td></tr><tr><td>TIP1;1, 1;2, 1;3, 2;1,<br>2;2, 2;3</td><td>CMV 1a</td><td>Cucumber mosaic<br>virus 1a</td><td>Y2H, SRS, co-IP</td><td>(133)</td></tr></tbody></table>

2416

2417

2418

2419

2420

2421

The table lists all plant aquaporin-interacting proteins that have been individually validated. The molecular function of each partner is indicated. The methods used to demonstrate a protein-protein interaction are also shown. FRET: Fluorescence Resonance Energy Transfer; FLIM: Fluorescence-Lifetime Imaging Microscopy; BiFC Bimolecular Fluorescence Complementation; Split-Ub: Split-Ubiquitin; Y2H: yeast two-hybrid. SRS: Sos Recruitment; co-IP: co-ImmunoPrecipitation
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

Maurel *et al.*, 2015
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_9.png" /><figcaption>Figure 7</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_10.png" /><figcaption>Figure 8</figcaption></figure>

Maurel *et al.*, 2015
A

Cell-to-cell
path

Apoplastic
path

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

AtPIP1;2

C

<figure><img src="image_13.png" /></figure>

AtPIP2;2

D

<figure><img src="image_14.png" /></figure>

AtPIP2;1

Figure 9
<figure><img src="image_15.png" /><figcaption>Figure 10</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_16.png" /><figcaption>Figure 11</figcaption></figure>

Maurel *et al.*, 2015
A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /><figcaption>Figure 12</figcaption></figure>

Maurel *et al.*, 2015
<figure><img src="image_20.png" /><figcaption>Figure 13</figcaption></figure>

Maurel *et al.*, 2015