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# Hormonal and environmental signaling pathways target membrane water transport

Christophe Maurel, Colette Tournaire-Roux, Lionel Verdoucq, Véronique Santoni-Kervella

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Christophe Maurel, Colette Tournaire-Roux, Lionel Verdoucq, Véronique Santoni-Kervella. Hormonal and environmental signaling pathways target membrane water transport. Plant Physiology, 2021, 10.1093/plphys/kiab373. hal-03440937

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Special issue on Transport and signaling

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**Short title:** Signaling pathways target membrane water transport

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**Corresponding author:** Christophe Maurel: christophe.maurel@cnrs.fr

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**Hormonal and environmental signaling pathways target membrane water transport**

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Christophe Maurel, Colette Tournaire-Roux, Lionel Verdoucq, and Véronique Santoni

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BPMP, Univ Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France.

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**ORCID IDs:** 0000-0002-4255-6440 (CM); 0000-0002-2966-5597 (C. T.-R); 0000-0001-8238-3700 (L.V.);

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0000-0002-1437-0921 (V.S.);

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**Materials distribution statement:**

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The author responsible for distribution of materials integral to the findings presented in this article in
accordance with the policy described in the Instructions for Authors

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(https://academic.oup.com/plphys/pages/General-Instructions) is:

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Christophe Maurel (christophe.maurel@cnrs.fr).
**One-sentence summary:** This paper describes the multiple signaling pathways targeting cell and tissue hydraulics to maintain the plant water status, and it reveals the commonalities of these pathways.

**Author contributions:** All authors contributed to the literature survey. CM wrote the article which was revised by all other authors. C. T.-R., L.V. and V.S. designed the figures and table.

**Funding information:** This work was supported in part by the Agence Nationale de la Recherche (ANR-18-CE92-0055)

## Introduction

As elaborate terrestrial living organisms, vascular plants have to maintain their water status as tightly as possible and throughout their life cycle (Davies and Bennett 2015; Steudle 1992). Particularly challenging processes include the expansion/growth of underground and aerial parts and the desiccation, imbibition and germination of seeds and pollen. Most often, these processes occur under variable environmental conditions, including changes to water availability and temperature in the soil and atmosphere, which have marked impacts on the plant water status. The latter is primarily maintained through tight stomatal regulation controlling transpirational water flow (Tardieu et al. 2015). Osmotic adjustment and regulation of water permeability (hydraulics), which both contribute to water potential gradients and water movement intensity, provide a complementary and finely-tuned regulation of plant water relations at the cell and tissue levels. In support for these general principles, a large body of studies has shown that plant water transport and its molecular components are responsive, across diverse time scales, to an extremely wide array of environmental and hormonal signals (Maathuis et al. 2003; Maurel et al. 2015; Chaumont et al. 1998; Aroca et al. 2012). Thus, plant water relations are under constant adjustment in virtually any physiological context.
Plant aquaporins, which are present in the plasma and intracellular membranes of most plant cells, play a central role in these processes by ensuring cell-to-cell water transport and to a lesser extent, single cell osmotic regulation (Maurel et al. 2015; Chaumont and Tyerman 2014; Tyerman et al. 1999). More generally, aquaporins have emerged as central membrane targets of environmental and hormonal signalling pathways acting on plant water relations. In addition, aquaporins are highly abundant proteins amenable to biochemical (proteomic) and cell biological approaches. Besides studies in the plant, their function and regulation can be easily studied after functional reconstitution (proteoliposomes) or heterologous expression (yeast, *Xenopus* oocytes). Thus, aquaporins are particularly relevant and convenient entry points for bottom up dissections of plant signalling cascades.

Due to the central position of aquaporins in multiple hormonal and environmental responses determining the plant water status, the first aim of the present review was to inventory corresponding signalling pathways. Beyond the diversity of these pathways, a knowledge network with common or converging paths is now emerging. Conversely, the dedicated study of signalling pathways per se seems to reveal regulation states/contexts that escaped classical physiological analyses of plant water relations. A particularly challenging question is to understand how plants are able to perceive and respond to combined stresses.

# 1) Water deficit, abscisic acid (ABA) and related signals

## 1.1 Drought.

### ABA-dependent pathways.

Whereas physiological links between drought stress, ABA and plant water transport have been identified for some time, it is only recently that the signalling mechanisms involved have started to be elucidated. Studies on epidermal peels from wild-type and aquaporin knock-out Arabidopsis (*Arabidopsis thaliana*) plants have revealed that the plasma membrane aquaporin Plasma Membrane Intrinsic Protein 2;1 (AtPIP2;1) is required for ABA-induced stomatal closure, but dispensable during
light- and CO₂-induced stomatal movements (Grondin et al. 2015). Consistent with this, isolated guard cell protoplasts responded to exogenous ABA by a two-fold increase in osmotic water permeability that was strictly dependent on the functionality of AtPIP2;1. This aquaporin, which is known to be permeable to H₂O₂ (Dynowski et al. 2008), was also required for ABA-induced accumulation of H₂O₂ in the guard cell interior (Rodrigues et al. 2017). These studies establish a dual water- and H₂O₂-transport function for guard cell plasma membrane aquaporins as players and targets of signalling processes triggering stomatal closure (Grondin et al. 2015; Rodrigues et al. 2017). More specifically, *in vitro* phosphorylation assays and transgenic plant analysis led to a model whereby Snf1-related protein kinase 2.6 (SnRK2.6)/Open stomata 1 (OST1), a protein kinase that plays a pivotal role in ABA signalling, is able to phosphorylate AtPIP2;1 at a specific cytosolic Ser residue (Ser121), with this phosphorylation being necessary for proper ABA-induced stomatal closure. SnRK2.6/OST1 also activates plasma membrane NADPH oxidases, which feed superoxide dismutases for production of apoplastic H₂O₂ (Maurel et al. 2016) (Figure 1A). While the above mentioned studies were performed on epidermal peels of wild-type and pip2;1 plants, we are concerned that intact leaves of a quadruple pip1;1 pip1;2 pip2;1 pip2;2 mutant showed normal responses to ABA (Ceciliato et al. 2019). We hypothesize that integration of guard cells within whole leaf physiology can compensate for the defects captured in isolated guard cells or protoplasts. We also note that, in contrast to stomata, ABA can down-regulate aquaporin activity in bundle-sheaths (Shatil-Cohen et al. 2011) and roots (Rosales et al. 2019). Whereas the canonical ABA signaling machinery formed by RCAR/PYR/PYL receptors, Protein Phosphatase 2C (PP2C) co-receptors acting on Class 2 Sucrose-Non-Fermenting Protein kinases (SnRK2s) is likely to be involved, in roots in particular (Rosales et al. 2019), the exact components providing these hydraulic regulations remain as yet unknown. Aquaporins and water transport can also be under ABA control through more indirect pathways. In Arabidopsis, ABA induced the expression of the membrane protein Tryptophan-rich sensory protein/translocator (TPSO), which physically interacts with AtPIP2;7 in the early secretory pathway, to trigger its degradation through the autophagic pathway, thereby reducing its
water transport activity at the plasma membrane (Hachez et al. 2014b). In rice (Oryza sativa), the gene encoding a leucine-rich repeat (LRR) receptor like kinase (RLK) named Leaf Panicle 2 (LP2) is down-regulated by drought and ABA, and acts a negative regulator of drought response (Wu et al. 2015). Its action involves a molecular interaction at the plasma membrane with the drought-responsive aquaporins OsPIP1;1, OsPIP1;3, and OsPIP2;3.

## ABA-independent pathways

Drought can also exert ABA-independent effects on aquaporin expression and function. For instance, several aquaporin gene promoters harbour Drought Responsive Elements (DRE) that promote the binding of DREB transcription factors (Rae et al. 2011). Other classes of transcription factors (e.g. AP2/EREBP, ASR1) also confer drought-dependent expression on aquaporin genes (Zhu et al. 2014; Ricardi et al. 2014). Furthermore, induction under severe drought of ubiquitin-conjugating (E2) enzyme UBC32 and RING-type E3 ligase, which both interact with AtPIP2;1 and AtPIP2;2, leads to ubiquitination and degradation of these aquaporins, thereby preventing their negative impact on drought tolerance (Chen et al. 2021).

A functional interaction between an aquaporin (RhPIP2;1) and a membrane tethered MYB protein named RhPTM was recently described in rose (Zhang et al. 2019b). In brief, dehydration stress induced C-terminal phosphorylation of RhPIP2;1 at Ser273, which in turn led to membrane release and nuclear activation of RhPTM. RhPTM itself acts as a transcription factor with general growth inhibitory effects. This landmark work establishes an unexpected link between hydraulics and a transcriptionally determined metabolic control of growth that promotes water deficiency tolerance. Yet, the protein kinases/phosphatases that act on Ser273 of RhPIP2;1 and their drought-dependent activation/inhibition mechanisms remain to be identified. Similarly, some PIP aquaporins of Arabidopsis (e.g. AtPIP2;4) show enhanced phosphorylation as early as 5 min after exposure to a hypertonic stress (0.3 M mannitol) (Stecker et al. 2014).

Cell biological approaches are now developed to address associated regulatory mechanisms. An improved super-resolution microscopic technique called single particle tracking Photo-Activated
Localization Microscopy (sptPALM) was used to monitor the lateral mobility of proteins at the plasma membrane (LTi6a, AtPIP2;1) and tonoplast (AtTIP1;1) (Hosy et al. 2015). In contrast to the other membrane proteins, AtPIP2;1 showed a low lateral mobility, was confined to membrane nanodomains and its mobility was enhanced upon a hyperosmotic stress. sptPALM further showed how reactive oxygen species (ROS) control specific diffusion and nano-organization of membrane cargo proteins (Martiniere et al. 2019). In particular, ROS initiated clustering of AtPIP2;1 and its removal from the plasma membrane. This process is achieved in part by clathrin-mediated endocytosis. It is linked to further aquaporin internalization and transfer into the endocytic pathways (Zwiewka et al. 2015). In the longer term, the same aquaporins are subjected to ubiquitination and further degraded (Lee et al. 2009) in a phosphorylation-dependent manner (Chen et al. 2021). In addition, a role of autophagic pathways was recently uncovered in *Medicago truncatula* under water deficit (Li et al. 2020b). A dehydrin (*MtCAS31*) was shown to act as a cargo receptor for the *MtPIP2;7* aquaporin. *MtCAS31* further promoted complex formation with the autophagy-related protein ATG8a to facilitate *MtPIP2;7* degradation. This, in turn, reduced water loss under water deficit and improved drought tolerance (Li et al. 2020b). The possible relation of this mechanism with the ABA-dependent TPSO pathway (Hachez et al. 2014b) mentioned above is not yet clear.

Finally, a bottom-up approach was used to further explore the signaling mechanisms acting upstream of aquaporins in the Arabidopsis root under hyperosmotic stress. A RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) pathway defined by two NADPH oxidases and an additional pathway involving apoplastic ascorbate and iron were found to mediate osmotic stress-induced ROS production (Martiniere et al. 2019). Furthermore, osmotic stress was shown to trigger interaction of a Rho GTPase, Rho-of-Plant 6 (ROP6) with the two NADPH oxidases (Smokvarska et al. 2020). This interaction occurs in specific plasma membrane nanodomains that are necessary and sufficient to transduce production of ROS and several plant adaptive responses to osmotic constraints (Figure 1B).

## 1.2 Salinity
Under salt stress, PIP aquaporins are subjected to cellular regulations that are reminiscent of those seen under a purely osmotic stress. In Arabidopsis roots for instance, a 100 mM NaCl treatment decreased C-terminal phosphorylation at Ser283 of AtPIP2;1 after 2-4 h (Prak et al. 2008). On a shorter timescale (30 min), a salt treatment also enhanced the lateral diffusion of AtPIP2;1 at the plasma membrane (Li et al. 2011) and its constitutive cycling (Luu et al. 2012). These movements lead to internalization of AtPIP2;1 from the plasma membrane, and its transfer to the pre-vacuolar and vacuolar compartments, a process that is mediated by ROS (Boursiac et al. 2008) (Figure 1B) and is dependent on clathrin, phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinase (PI4K)(Ueda et al. 2016). These mechanisms may well be generalized: AtPIP2;7, another major aquaporin isoform in the Arabidopsis root, showed a combined transcriptional down-regulation and protein internalization that resulted in a dramatic decrease in cell and tissue hydraulics (Pou et al. 2016). As in the case of osmotic stress, the primary signalling events triggering salt-dependent aquaporin regulation are not elucidated. Yet, salt-dependent expression of AtPIP2;3 and AtPIP2;5 is in part mediated by SnRK2s of subclass 1 (Kawa et al. 2020). Some of these protein kinases (SnRK2.4, SnRK2.10) interact with and phosphorylate VARICOSE (VCS) and VARICOSE RELATED (VCR) (Kawa et al. 2020; Soma et al. 2017), two components of the mRNA decapping complex that catalyzes the first step of mRNA decay.

A role of PIP aquaporins in ion transport, tentatively associated to the so-called Non Selective Cation Channels described by patch clamp in many cell types, was recently proposed in Arabidopsis and barley (Tran et al. 2020; Byrt et al. 2017). As thoughtfully discussed by (Tyerman et al. 2021), this activity may well be central during plant response to salinity stress. The ionic conductance of AtPIP2;1 is repressed by calcium and strongly enhanced upon C-terminal phosphorylation (Qiu et al. 2020). In barley, HvPIP2;8 shows similar regulation, and, in addition, its gene is induced in shoots of salt stressed plants, specifically in a tolerant cultivar (Tran et al. 2020). The connections of these regulatory processes to upstream salt stress signaling events and, possibly, to water transport regulation remain to be investigated.
## 2) Other abiotic constraints and stimuli

### 2.1 Oxygen availability

Flooding stress, whether due to water logging or submergence, deeply impacts plant water relations by favoring a massive inflow of water through roots while transpiration is directly or indirectly impaired (Tan et al. 2018). Two pathways leading to aquaporin regulation have been uncovered in this context (Maurel and Nacry 2020). On the short term, a pronounced cytosolic acidosis can be observed under oxygen (O₂) deprivation due to the metabolic production of organic acids while a drop in cellular respiration and ATP production hampers efficient proton (H⁺) extrusion by H⁺-ATPases. This in turn induces protonation of a His residue located on the second cytosolic loop of PIPs, triggering channel gating. It is remarkable that this His residue is perfectly conserved in PIPs of all species, supporting the generality of the H⁺-dependent gating mechanism (Tournaire-Roux et al. 2003). We note that certain plant species do not show any effect of hypoxia on root hydraulic conductivity (Lpᵣ). In the case of two lupin species (*Lupinus angustifolius*, *Lupinus luteus*), hypoxia induced a marked inhibition of cortical cell hydraulic conductivity but had no substantial effect at the whole root level, suggesting the predominance of an apoplastic pathway during water uptake (Bramley et al. 2010).

The second pathway was recently uncovered through quantitative genetic analysis of Arabidopsis root hydraulics. It is under the control of Hydraulic Conductivity of Root 1 (HCR1) (Shahzad et al. 2016). HCR1 mediates root responses to hypoxia in relation to potassium (K⁺) availability, thereby acting as a negative regulator of Lpᵣ and root aquaporins. HCR1 encodes a RAF-type MAP3K protein kinase that specifically accumulates in the presence of K⁺ and absence of O₂. HCR1 enhances the abundance of RAP2.12, a key Ethylene Response Factor (ERF) VII transcription factor that is stabilized under O₂ starvation and triggers the core anaerobic transcriptional response. We have proposed that HCR1 phosphorylates RAP2.12 or one of its direct regulators in planta and
thereby potentiates the most early plant responses to O₂ starvation (Shahzad et al. 2016). However, the mode of aquaporin down-regulation through this pathway has not been elucidated.

## 2.2 Nitrogen (N)

Nitrate (NO₃⁻) is the major inorganic N source absorbed by non-wetland plants and its uptake and metabolism are tightly associated with water utilization. Whereas ammonium has a rather negative effect on water absorption, NO₃⁻ was shown to enhance Lpᵣ in multiple plant species. Although post-translational regulation may also be involved, the increase in Lpᵣ induced by NO₃⁻ was shown to correspond to up-regulation of aquaporin expression (Gorska et al. 2008; Gloser et al. 2007). It has also been disputed whether NO₃⁻ acts directly or through its assimilation products. In Arabidopsis, the conserved Lpᵣ response of a nitrate reductase mutant (nia) supports the first hypothesis (Li et al. 2016). Furthermore, characterization of various Arabidopsis genotypes, altered in NO₃⁻ transport, assimilation, or signaling and growing under various N supply conditions revealed a strong positive relationship between Lpᵣ and NO₃⁻ accumulation, but in shoots rather than in roots (Li et al. 2016). This suggests a control of Lpᵣ by putative long-distance signals generated in the shoots. Such systemic regulation of aquaporins was also observed in split-root experiments with rice plants subjected to heterogeneous NO₃⁻ treatments (Ishikawa-Sakurai et al. 2014). We also note that Lpᵣ regulation by transpiration or wounding (Laur and Hacke 2013; Ishikawa-Sakurai et al. 2014; Liu et al. 2014; Vandeleur et al. 2014) may also be mediated by shoot-borne signals acting on root hydraulics.

Although NRT1.1 is considered as the major NO₃⁻ sensor, the high-affinity nitrate transporter NRT2.1 is also thought to have a signaling role. In contrast with *nrt1.1* plants, *nrt2.1* plants showed a pronounced reduction in Lpᵣ, by up to 30% (Li et al. 2016). Surprisingly, this regulation was independent of NO₃⁻ and hence, of NRT2.1 expression level. Thus, the signalling mechanisms involved in this newly discovered pathway remain fairly mysterious.

## 2.3 Cold
214 Both moderate cold and freezing challenge the plant water status. For instance, low temperature,
215 which reduces Lp<sub>r</sub> due to a higher viscosity of water and aquaporin down-regulation, causes water
deficit and growth arrest in leaves (Lee et al. 2012). In maize, aquaporins seems to play a key role in
216 the recovery of Lp<sub>r</sub> that is specifically observed in chilling tolerant genotypes (Aroca et al. 2005).
217 Similarly, cold acclimation of Arabidopsis seedlings is accompanied by upregulation of two specific
218 PIP genes (AtPIP1;4; AtPIP2;5) (Rahman et al. 2020), which also show mRNA accumulation in leaves
219 under water deficit (Alexandersson et al. 2005). In rice, proper expression and function of Calcium-
220 dependent Protein Kinase (CDPK) OsCPK17 appears to be key for plant adaptation to low
temperature stress (Almadanim et al. 2017). A comparative phosphoproteomic approach further
221 identified OsPIP2;1 and OsPIP2;6 as putative targets of OsCPK17. A direct activation by OsCPK17 is
222 further supported by *in vitro* assays showing that the two aquaporins can be phosphorylated by the
223 protein kinase in a calcium-dependent manner. Since OsCPK17 also targets sugar metabolism, it was
224 proposed that cold stress-activated OsCPK17 phosphorylates OsPIP2;1 and OsPIP2;6 to coordinate
225 membrane water transport with cell osmotic regulation (Almadanim et al. 2017).
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228 **<u>2.4 Circadian control.</u>**

229 The circadian clock allows the fine tuning and synchronization of key physiological, metabolic and
230 signaling pathways, in phase with daily variations of environmental parameters. While circadian
231 regulation of tissue hydraulics has been largely documented (Henzler et al. 1999), it is only recently
232 that the corresponding modes of aquaporin regulation have been investigated. In roots of
233 Arabidopsis and maize, the control of aquaporin expression seems to be largely transcriptional and
234 depends on well-identified clock components such as ELF3 (Takase et al. 2011; Caldeira et al. 2014).
235 In maize, the oscillation amplitude of aquaporin expression and plant hydraulic conductance was
236 strongly enhanced under water deficit, while oscillating expression of the core circadian clock genes
237 was not altered (Caldeira et al. 2014). Interestingly, this specific potentiation of the hydraulic
238 outputs, which possibly depends on ABA, appears to be beneficial for cumulated plant growth under
water deficit whereas steady tissue hydraulic properties are more appropriate under water replete conditions. These data show how adaptive responses of plant hydraulics to specific environmental variations rely on exquisite coupling to endogenous signalling mechanisms.

In contrast with roots, the circadian control of leaf hydraulics seems to be mediated through post-translational modifications of proteins (Prado et al. 2019; Choudhary et al. 2015). In particular, the rosette hydraulic conductivity ($K_{ros}$) of Arabidopsis shows circadian oscillations with a peak at midday, in phase with quantitative variations of PIP2 aquaporin phosphorylation (Prado et al. 2019). Remarkably, this modification is necessary but not sufficient for circadian control of $K_{ros}$, which requires the additional contribution of 14-3-3 proteins (Prado et al. 2013; Prado et al. 2019). Two of these (GRF4, GRF10) can physically interact with AtPIP2;1 and modulate its function in *Xenopus* oocytes depending on the phosphorylation status of the aquaporin C-terminal tail, with more pronounced effects on phosphomimetic than on phosphodeficient forms (Prado et al. 2019). Much remains to be learned about the protein kinase/protein phosphatase machinery that ensures oscillations in aquaporin phosphorylation and, possibly, a synchronized activation of 14-3-3 proteins.

## 3) A multiplicity of endogenous signals

### 3.1 Auxin.

Transcriptomic analyses in Arabidopsis have revealed a dramatic down-regulation of aquaporin expression during lateral root formation (Péret et al. 2012). This effect is mediated in part by auxin which, more generally, down-regulates most aquaporin expression in roots within 5-10 hours, and, consequently, $Lp_r$. Auxin also induces, after 24h, a drop in turgor of cortical cells. Interestingly, mutant analyses have shown that all these molecular and physiological responses are mediated through Auxin Response Factor 7 (ARF7), a transcription factor that serves as a master regulator of lateral root formation and emergence. Yet, the down-stream targets of ARF7 in these responses remain to be identified. Our current understanding is that a finely tuned spatial and temporal control of PIP expression favours water entry into the lateral root primordium. This inflow is made at this
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expense of overlaying cells, thereby reducing their mechanical resistance and facilitating lateral root emergence.

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## 3.2 Methyl jasmonate (MeJa)

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Contrasting effects of MeJa on root water transport have been reported by different groups. (Sanchez-Romera et al. 2014) observed that exogenously supplied MeJA increases $L_{p,r}$ in bean, tomato and Arabidopsis. These effects are associated with an increased phosphorylation of PIP2s and partly depend on ABA, which accumulates in response to MeJa. In bean, the fluoridone inhibitor counteracted the effects of MeJA on both ABA accumulation and $L_{p,r}$. By contrast, exogenous MeJa induced a decrease in cell hydraulic conductivity in the cortex of Arabidopsis roots (Lee and Zwiazek 2019). Surprisingly, *fad3-2* and *fad7-2* plants, which are defective in MeJa endogenous biosynthesis, were insensitive to this external treatment. These contrasting results suggest that effects of MeJa on root hydraulics are cell-type specific, somewhat indirect and involve important hormonal cross-talks that remain to be uncovered.

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## 3.3 Ethylene

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The effects of ethylene on aquaporin function in various plant species and organs have revealed the contribution of multiple signalling pathways. In rose, ethylene acts as a repressor of flower opening. It negatively controls petal cell expansion through down-regulation of *RhPIP2;1* (Ma et al. 2008) and *RhPIP1;1* (Chen et al. 2013), which functionally interacts with the former. Consistent with these negative hormonal effects, silencing of ethylene receptors (*RhETRs*) which, in the absence of the hormone function as repressors of ethylene effects, resulted in an ethylene-independent decrease in *RhPIP1;1* expression (Chen et al. 2013). In Arabidopsis leaves, ethylene exerts positive effects on water transport through enhanced C-terminal phosphorylation of *AtPIP2;1* (Qing et al. 2016). These post-translational effects require the ETR-1 receptor and its interacting partner Constitutive Triple-Response 1 (CTR1). They are independent of the major transcriptional pathway involving Ethylene-Insensitive 1 (EIN1) and Ethylene-Insensitive 3-like (EIL3), which would in turn mediate the

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transcriptional down-regulation of PIPs, as mentioned above. Tentative regulatory networks acting
on aquaporin phosphorylation, under the control of ethylene and through balanced effects on
protein kinases and phosphatases, were inferred in part from a differential phosphoproteomics
approach in Col-0 and *ctr1* plants (Yang et al. 2013). Since ethylene enhances auxin accumulation,
hormonal interactions will also have to be taken into account to comprehend the multiple pathways
leading to ethylene-dependent aquaporin regulation (Yang et al. 2013; Qing et al. 2016)

## 3.4 Sugars

Sugars act as metabolites but also signalling molecules to regulate various whole plant and cellular
processes such as photosynthesis and stomatal movements. With respect to tissue water transport,
sucrose was shown to enhance Arabidopsis *Lp*<sub>r</sub> specifically in the dark, whereas it inhibited *Lp*<sub>r</sub> in the
light, as did other osmotica (Di Pietro et al. 2013). Sucrose also induced changes in AtPIP2;6
phosphorylation (Niittylä et al. 2007), thereby providing a tentative link between light, sugars and
aquaporins. This link was corroborated by the high negative correlation existing between the
expression of aquaporins and either exogenous glucose or the expression of Hexokinase 1 (AtHXK1)
in over-expressing or knock-out transgenic Arabidopsis (Kelly et al. 2017). Protein kinases that are
involved in sucrose-induced phosphorylation responses, among which RLKs (e.g. SIRK1) and receptor-
like cytoplasmic kinases (RLCKs e.g. Brassinosteroid Signaling Kinase 8 (BSK8)) have been identified
through typical changes in their phosphorylation status within the few minutes following sucrose
resupply. Some aquaporins were subsequently identified as downstream targets of these protein
kinases (Wu et al. 2019; Wu et al. 2013; Wu et al. 2014). For instance, molecular interactions of SIRK1
with AtPIP2;6 were established through pull down experiments and phosphorylation assays using an
AtPIP2;6-specific peptide. The mesophyll protoplast water permeability of Arabidopsis lines showing
different expression levels of active SIRK1 revealed that the RLK directly regulates aquaporins via
phosphorylation in a sucrose dependent manner (Wu et al. 2013). In addition, SIRK1 forms a complex
with its co-receptor QSK1. SIRK1 shows autophosphorylation, subsequently trans-phosphorylates
QSK1, which in turn enhances and stabilizes the interaction of aquaporins with the receptor kinase complex (Wu et al. 2019). Our understanding of BSK8 action on aquaporins is not as precise, but the RLCK possibly acts on another isoform, AtPIP2;7, that shows reduced phosphorylation in a bsk8 mutant. Although still incomplete, the whole set of data suggests intricate osmotic, metabolic and signalling effects of sugars acting on aquaporins through multiple receptor pathways and time frames (Wu et al. 2014). At the whole plant level, the somewhat opposite effects of sugar on hydraulics depending on organs, timing, or environmental conditions surely reflect contrasting contexts where maintenance of growth or of water content dominate one another.

## 3.5 Peptidic hormones and other unknown ligands

The endodermis functions as a major barrier within the root, determining fundamental features of water and nutrient absorption from the soil (Doblas et al. 2017). These properties follow from the typical differentiation of endodermal cells, with an initial formation of Casparian strips made out of lignin and further deposition of suberin lamellas that wrap the whole cell. Casparian strips mostly block apoplastic solute movements and favor xylem loading by preventing solute backflow from the stele to the cortex. Suberin lamellas, rather, limit transcellular transport of nutrients and possibly water at the endodermis. These barriers, which are at the cross-roads of mineral nutrient and water uptake, are therefore under tight control by endogenous and environmental factors (Barberon et al. 2016). For instance, the loss of Casparian strip integrity is detected by diffusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 (CIF1 & 2) which act through the SCHENGEN3 receptor-like kinase (Pfister et al. 2014, Doblas et al. 2017, Nakayama et al. 2017) to rebalance water and mineral nutrient uptake. More specifically, CIF1 and CIF2 induce a reduction in Lp, driven by the deactivation of aquaporins (Wang et al. 2019). They also limit ion leakage through deposition of suberin in endodermal cell walls. Inhibition of aquaporins seems to be a primary effect of CIFs, which in turn may promote suberin deposition. Interestingly, CIF1 and CIF2 act independently of ABA signaling, which can also be a potent regulator of aquaporins in endodermal cells.
339 Casparian strip deposition is guided by the local expression of a family of CAsparian Strip
340 domain Proteins (CASPs). Related proteins named CASP-like are expressed throughout the plant.
341 Four of these (CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were identified as interacting with
342 AtPIP2;1 (Bellati et al. 2016). Although the physiological relevance of these molecular interactions is
343 not known, CASPL1B1 was shown to specifically activate phosphorylated AtPIP2;1 (Champeyroux et
344 al. 2019), pointing to a putative composite regulatory pathway.

345 **4) Biotic interactions**
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346 **4.1 Plant immunity**
---

347 In recent years, several landmark works have reported a crucial role of aquaporins in plant defense
348 (Zhang et al. 2019a; Li et al. 2020a; Wang et al. 2018; Tian et al. 2016; Ai et al. 2021). AtPIP1;4 was
349 identified as one of the PIPs, the expression of which is induced by infection with *Pseudomonas
350 syringae* pv. *Tomato* (Tian et al. 2016). The use of intracellular and extracellular ROS dyes in yeast
351 and transgenic Arabidopsis further revealed the capacity of AtPIP1;4 to mediate the diffusion of
352 apoplastic H2O2 into the cytoplasm. This process represents a major step in the signaling response of
353 plants to pathogen-associated molecular patterns (PAMPs), which activate RBOH NADPH oxidases
354 present on the plasma membrane. More generally, analysis of knock-out mutants and overexpressing
355 plants revealed that AtPIP1;4 is necessary for PAMP-triggered immunity (Tian et al. 2016) and
356 mediates systemic acquired resistance (SAR) through a parallel pathway involving activation of
357 NONINDUCER OF PR GENES1 (NPR1). These findings establish a major signaling role of PIPs in plant
358 defense (Figure 1C). Interestingly, a mechanism allowing bacterial effectors to counteract this
359 aquaporin signalling function was recently uncovered (Ai et al. 2021). In brief, a Crinkler effector
360 (CRN78) from *Phytophthora* displaying a protein kinase activity was shown to phosphorylate the C-
361 terminal tail of several H2O2-transporting PIPs of soybean or *Nicotiana benthamiana*, thereby
362 triggering their degradation through a proteasome-dependent pathway. This led to inhibition of
363 H₂O₂-accumulation and reduced immunity of these plants when infected by *Phytophthora oomycetes*
364 (Ai et al. 2021).

365 A signalling role for aquaporins during plant defense was also uncovered for AtPIP2;1 in
366 guard cells, with this aquaporin being necessary for flg22-induced stomatal closure. In this case, a
367 pathway was proposed, whereby the bacterial PAMP flg22 binds to its receptor FLS2 leading to the
368 successive activation of the BAK1 co-receptor and SnRK2.6 protein kinase. This in turn
369 phosphorylates AtPIP2;1 at Ser121 to promote both water and H₂O₂ transport as shown in the
370 context of ABA-induced stomatal closure (Rodrigues et al. 2017) (Figure 1A). Flagellin (flg22) also
371 enhanced lateral diffusion of AtPIP2;1 at the guard cell plasma membrane through effects on
372 microtubules, resulting in its internalization to the cell (Cui et al. 2021). It is not yet clear whether
373 these effects contribute to the primary role of AtPIP2;1 in stomatal closure or reflect a feedback
374 inhibition of its function due to flg22-induced ROS accumulation. Interestingly, AtPIP2;1 dynamics at
375 the plasma membrane of adjacent (subsidiary) cells showed a distinct dependency on the
376 cytoskeleton and were somewhat insensitive to flg22 (Cui et al. 2021). Thus, cell-specific regulation
377 of aquaporins probably contributes to an integrated control of water exchange between cell types
378 during stomatal movements.

379 PAMPs such as chitin are produced during plant attack by fungi and insects. Aquaporin-
380 mediated water transport in leaf tissues such as the mesophyll and bundle sheath was shown to be
381 down-regulated by chitin (Attia et al. 2020). This inhibition could contribute to plant defense by
382 hampering vascular pathogen propagation in xylem vessels and by promoting stomatal closure
383 through long-distance hydraulic signalling. Although the mode of aquaporin regulation by chitin
384 remains as yet unknown, the contribution of two chitin receptor-like kinases, chitin elicitor receptor
385 kinase 1 (CERK1) and lysine motif receptor kinase 5 (LYK5), could be established (Attia et al. 2020)

386 Recent work also points to another molecular role of aquaporin during bacterial infection.
387 For instance, rice OsPIP1;3 and Arabidopsis AtPIP1;4 molecularly interact with the bacterial protein
388 harpin (Hpa1) which is secreted by Gram-negative pathogenic bacteria. Thus, PIPs serve as receptors
389 for harpins at the plant plasma, which in turn mediate the delivery of effector proteins (such as 390 PthXo1) into the plant cell cytoplasm (Li et al. 2019; Li et al. 2015b). Along the same lines, AtPIP2;7 391 was recently shown to form a molecular complex with the protease domain of the tobacco etch 392 potyvirus Nla protein (Martinez et al. 2016). Due to its numerous additional connections to stress 393 responses, the aquaporin seems to be a preferential target during viral infection. 394

395 The overall data show that aquaporins are at the heart of the arms race between plants and 396 their aggressors. On the one hand, aquaporins help plants resist pathogens by contributing to 397 defense signaling pathways and by preventing their ingress through promotion of stomatal closure 398 (Zhang et al. 2019a; Li et al. 2020a). On the other hand, some aquaporins can be hijacked by bacterial 399 or viral pathogenic machineries, thereby promoting pathogen propagation. 400

401 **4.2 Myccorhizae** 402

400 Aquaporins also contribute to positive plant-microbe interactions whereby they play important roles 401 in nutrient transport and abiotic stress responses. In particular, arbuscular mycorrhizal symbiosis can 402 notably improve the plant water status. A fine dialog exists between the fungus and the host plants, 403 leading to precise transcriptional and post-translational regulation of plant aquaporins, particularly 404 under drought (Quiroga et al. 2019). Similarly, the sub-cellular expression of specific tonoplast 405 aquaporins seems to be subtly targeted in N-fixing symbiotic nodules (Gavrin et al. 2014). Although 406 strong hormonal interplays are suspected, the signaling pathways leading to these regulations are as 407 yet unknown. 408

5) **Commonalities of signalling pathways**
***

409 The survey above provides a broad but scattered knowledge of signalling processes targeting cell and 410 tissue hydraulics in plants (Table 1). Yet, a few main principles do emerge from this information.

411 Whereas transcriptional control is definitely key for long-term adjustment of plant hydraulics 412 in response to environmental or developmental cues, post-translational mechanisms seem to 413 dominate short term (min and hours) regulations, as these are more tailored for fast and reversible
414

responses to the multiple factors (including water availability) acting on the plant water status. Here,
we discuss a few common players of the latter regulatory mechanisms.

415

416

Firstly, ROS play a central signalling role in such regulation of plant water transport (Figure 1).
They are produced in response to numerous stimuli including osmotic, salt and cold stress, nutrient
deprivation and hormones (salicylic acid, ABA). Although several ROS generating systems may
operate at the plasma membrane (Martiniere et al. 2019), RBOH NADPH oxidases seem to occupy a
central position in this device (Li et al. 2011; Martiniere et al. 2019). By enhancing constitutive cycling
of aquaporins at the plasma membrane and shifting the equilibrium towards endocytosis, ROS
promote aquaporin internalization, thereby reducing their density at the cell surface and, as a
consequence, cell hydraulic conductivity (Boursiac et al. 2008; Wudick et al. 2015). As suggested by
recent work in guard cells (Cui et al. 2021), this mechanism could provide a feedback regulation in
contexts where aquaporins contribute to the initial steps of ROS signalling.

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

The sub-cellular trafficking and regulated degradation of aquaporins also appears to be a
main component of plant responses to environmental stimuli. Syntaxins and other traffic proteins,
such as adaptor proteins (AP), play well-established roles in these processes (Besserer et al. 2012;
Hachez et al. 2014a; Pertl-Obermeyer et al. 2016). While some internalized aquaporins accumulate in
pre-vacuolar compartments (Luu et al. 2012), a stress-response pathway under the control of
Tryptophan-rich sensory protein/translocator (TPSO) (Hachez et al. 2014b) can relay the above
mentioned trafficking to promote aquaporin degradation. The autophagic pathways described in
*Medicago truncatula* under water deficit (Li et al. 2020b) may also operate under other stressing
conditions. In addition, two recent works have pointed to a control by phosphorylation of ubiquitin-
dependent degradation of aquaporins in plants under abiotic or biotic stress (Ai et al. 2021; Chen et
al. 2021). This mechanism, which allows control of aquaporin abundance, further expands the
multiple, and possibly opposite, roles of phosphorylation in controlling aquaporin function.

439

In relation to the central role of phosphorylation, aquaporins are known to be targeted by
multiple classes of protein kinases. This feature holds promises to further decipher aquaporin
440 signalling pathways in bottom-up approaches (Figure 2). CDPKs have long been proposed as major
441 players of aquaporin regulation (Johnson and Chrispeels 1992; Johansson et al. 1996). Yet, there are
442 very few cases where their role was formally established (Ji et al. 2017; Almadanim et al. 2017). We
443 also note that CDPKs can be involved in upstream steps and therefore indirectly act on aquaporin
444 regulation (Li et al. 2015a). Due to their key role during stomatal movements (Yip Delormel and
445 Boudsocq 2019), it will be worth exploring the action of CDPKs on guard cell aquaporins in closer
446 detail.

447 SnRK2s represent another potentially important class of protein kinases acting on plant
448 aquaporins. Besides SnRK2.6 (OST1), which mediates ABA- and flg22-dependent activation of
449 AtPIP2;1 in guard cells (Grondin et al. 2015; Rodrigues et al. 2017), other SnRK2s may delineate
450 additional ABA- or osmotic-stress dependent pathways acting on aquaporins. In support for this,
451 functional expression of plant signalling pathways in yeast using a transcriptional readout indicated
452 that four SnRK2s initially thought to be specific for osmotic responses can also contribute to ABA
453 signalling (Ruschhaupt et al. 2019). Thus, the model of ABA-dependent regulation of AtPIP2;1 may be
454 extended to additional pairs of SnRK2s and PIPs.

455 RLKs constitute another major a class of serine/threonine kinases that perceive
456 environmental and extracellular developmental signals and transduce them via their intracellular
457 kinase domain. While their role in aquaporin regulation in the endodermis (SGN3) and during sugar
458 response (SIRK1, BSK8) can serve as models, we anticipate a role for RLKs in many other cellular
459 contexts including water stress (see Outstanding questions). For instance, a RLK (LP2) and a
460 Cytoplasmic Receptor-like Kinase (OsRLCK311), both from rice, were shown to interact with PIPs in
461 vivo (Wu et al. 2015; Sade et al. 2020), but the functional role of these interactions was not
462 definitively elucidated. Moreover, Arabidopsis RKL1, the activating signals of which remain to be
463 identified, was shown to molecularly interact with AtPIP2;1 (Bellati et al. 2016). Co-expression of
464 AtPIP2;1 with RKL1 in *Xenopus* oocytes resulted in enhanced AtPIP2;1 water transport activity by an
465 as yet unknown mechanism (Bellati et al. 2016). RLKs may also regulate aquaporins by non-canonical
mechanisms. For instance, co-expression of FERONIA and *AtPIP2;1* in *Xenopus* oocytes leads to inactivation of the latter (Bellati et al. 2016). This inactivation seems to require the protein kinase activity of FERONIA and is favored by two C-terminal phosphorylation sites of *AtPIP2;1* that, in contrast, have been described as activating sites. Thus, these phosphorylated residues may favor the FERONIA-*AtPIP2;1* interaction, which in turn would lead to aquaporin inhibition.

Finally, CBL-interacting protein kinases (CIPKs) represent major regulators of plant nutrition and response to abiotic stresses (Dong et al. 2020). It is rather surprising that, despite efforts from several laboratories including ours, no aquaporin has ever been identified as one of their numerous substrates. In addition, protein phosphatases, which surely play a key role in phosphorylation-dependent regulation of aquaporins, will deserve specific attention in future studies.

Lipid signalling represents another emerging component targeting aquaporins (see Outstanding questions). For instance, genetic alteration of phosphoinositides (phosphatidyl inositol [4,5] bisphosphate: PtdInsP2) in tobacco (*Nicotiana tabacum*) increased the osmotic water permeability and aquaporin activity of protoplasts isolated from suspension cells (Ma et al. 2015). The lipid effects may operate through direct interaction with aquaporins or through indirect cellular effects of PtdInsP2. For instance, interaction of PtdInsP2 with TPSO is required for interaction with AtPIP2;7 and its subsequent degradation (Jrkiewicz et al. 2020). A role for lipid signaling in PIP regulation is also suggested by the physical interaction between AtPIP2;1 and two phospholipases D (PLDδ and PLDγ1) (Bellati et al. 2016). In plants, PLDs and their enzymatic product phosphatidic acid (PA) play key roles in cellular responses to hormonal and abiotic stimuli. Along these lines, AtPIP2;1 and AtPIP2;2 were identified as PA-binding proteins (McLoughlin et al. 2013) but the functional effects of these interactions remain to be elucidated.

In complement to these targeted studies, we believe that systems biology approaches will help uncover previously unidentified signalling pathways targeting aquaporins. For instance, protein interaction networks involving aquaporins have pointed to links with clusters associated with brassinosteroid signaling (Bellati et al. 2016). A genome-wide search for membrane-protein
complexes has revealed other clusters that are enriched in aquaporins and have potential roles in stimulus-dependent regulation of water transport (Jones et al. 2014). Finally, a close inspection of aquaporin gene position in gene regulation networks will for sure reveal other modes of developmental or environmental regulation of aquaporins (Alexandersson et al. 2010).

## 6) Perspectives

Whereas this overview principally deals with chemical signals, whether abiotic or hormonal, there is still much to be learned about how physical cues lead to aquaporin regulation (see Outstanding questions). Particularly challenging seems the direct link between tissue hydraulics and transpiration that may well be mediated through hydraulic signals, or in other words, pressure-dependent regulation of aquaporins (Pou et al. 2013; Laur and Hacke 2013). Such dependency may also be at the basis of wound-induced alterations in root or leaf hydraulic conductivity (Vandeleur et al. 2014; Liu et al. 2014). Plant defence is another emerging context in which aquaporin regulation will have to be investigated. While the plant water status is crucial for resilience of plants under attacks, it is also a key target for pathogens to facilitate their proliferation within the plant (Xin et al. 2016). We now have some ideas on how aquaporins serve as both signalling components and targets during innate immunity (Zhang et al. 2019a). In contrast, the action of effectors on aquaporins and possible resistance mechanisms associated to these are just emerging (Ai et al. 2021).

Beyond regulation of water transport in a strict sense, we realize that mechanisms targeting other aquaporin transport functions may have some relevance for plant water relations or share similarities with mechanisms targeting the latter. For instance, aquaporin-mediated CO₂ transport is regulated through interaction with βCA4 carbonic anhydrase (Wang et al. 2016), indicating that the various molecular functions of aquaporin interactants and their ability to associate aquaporin regulation to upstream signalling cascades still represent a gold mine of information. Also, the ion transport of AtPIP2;1, which is strongly dependent on divalent cations and pH (Byrt et al. 2017), confirms that both Ca²⁺ and H⁺ are definitely crucial second messengers acting on aquaporin activity.
517

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525

Because plants are continuously exposed to combinations of environmental constraints, we realize that our analytic representation of single signalling pathways remains largely inappropriate. Although we emphasized how conserved pathways and their cross talks ultimately act on aquaporins, our knowledge of these processes remains very poor. More generally, basic information on how plant water transport is adjusted in response to combined stresses is still lacking. This large field of investigation, combining fundamental plant physiology and systems biology, is now necessary to properly comprehend the amazing capacity of plants to acclimate to a large array of stressful environments.
526

### Advances.

527

* ABA-dependent and independent signalling pathways act on aquaporins in plants under water deficit.

528

529

* Aquaporin-mediated water transport is targeted by multiple abiotic, biotic, or endogenous stimuli; recently identified stimuli include peptide hormones and PAMPs.

530

531

* ROS play a central signalling role in regulating water transport in plants by acting on aquaporin subcellular dynamics, localization and/or phosphorylation.

532

533

* Plasma membrane aquaporins exhibit a dual water- and $H_2O_2$-transport function, acting as players in and targets of signalling processes.

534

535

* Phosphorylation of aquaporins, which interferes with their gating, trafficking, and stability, is a major target of plant signalling cascades, and multiple classes of protein kinases that act on distinct phosphorylation sites have been identified.

536

537

538

539

### Outstanding questions.

540

* Is aquaporin-mediated $H_2O_2$ transport a common process in plant cell signaling?

541

* Which cellular signals trigger RLK-dependent aquaporin regulation?

542

* What is the role of lipid signaling in water transport regulation?

543

* What are the mechanisms for coupling physical stimuli to aquaporin regulation?

544

* How do aquaporins contribute to plant defense? What are their active roles in plant immunity, and how can they be hijacked by pathogens?

545

546

* What are the effects of combined stresses on plant water transport, and which signaling pathways are involved?

547

548
549 **Acknowledgments.** The authors thank their colleagues of the Aqua team for fruitful discussions. This

550 work was supported in part by the Agence Nationale de la Recherche (ANR- 18-CE92-0055).
<table><caption>**Table 1:** Molecular signaling mechanisms involved in aquaporin regulation under abiotic and biotic stresses and by endogenous signals</caption><thead><tr><th>Type of signal</th><th>Stimulus</th><th>Signalling intermediate</th><th>Molecular mechanism on aquaporins</th><th>Aquaporin regulation</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">ABIOTIC</td><td>drought (ABA-dependent)</td><td>SnRK2.6<br>LP2</td><td>AtPIP2;1 loop B<br>phosphorylation<br>interaction with OsPIP1;1,<br>OsPIP1;3 and OsPIP2;3</td><td>up-regulated activity<br>down-regulated activity</td><td>Grondin et al., 2015; Rodrigues et al., 2017<br>Wu et al., 2015</td></tr><tr><td>drought (ABA-independent)</td><td>RhPTM<br>TSPO<br>MtCas31/<br>MtATG8</td><td>interaction with RhPIP2;1<br>nd<br>MtCas31 as a cargo receptor<br>for MtPIP2;7</td><td>nd<br>AtPIP2;7 protein degradation<br>protein degradation by<br>autophagy</td><td>Zhang et al., 2019<br>Hachez et al., 2014b<br>Li et al., 2020b</td></tr><tr><td>osmotic</td><td>nd<br>RBOHs and<br>ROP6<br>clathrin<br>Rma1H1<br>UBC32</td><td>increased C-ter<br>phosphorylation of AtPIP2;4<br>membrane lateral mobility<br>nd<br>AtPIP2;1 ubiquitination<br>AtPIP2;1 ubiquitination</td><td>nd<br>clustering in nano-domains<br>protein internalization<br>protein degradation<br>protein degradation</td><td>Stecker et al., 2014<br>Hosy et al., 2015; Martiniere et al., 2019; Smokvarska et al., 2020<br>Zwiewka et al., 2015<br>Lee et al., 2009<br>Chen et al., 2021</td></tr><tr><td>salinity</td><td>clathrin,<br>PI3K, PI4K,<br>SnRK2.4,<br>SnRK2.10</td><td>C-ter phosphorylation of<br>PIP5; enhanced mRNA decay</td><td>protein internalization and<br>down-regulated activity</td><td>Prak et al., 2008; Boursiac et al., 2008; Ueda et al., 2016; Pou et al., 2016; Kawa et al., 2020</td></tr><tr><td>oxygen</td><td>cytosolic<br>protons</td><td>His protonation of PIPs</td><td>down-regulated activity</td><td>Tournaire-Roux et al., 2003</td></tr><tr><td>availability</td><td>HCR1</td><td>nd</td><td>down-regulated activity</td><td>Shahzad et al., 2016</td></tr><tr><td>Nitrogen</td><td>NRT2;1</td><td>nd</td><td>enhanced gene expression,<br>protein accumulation and<br>up-regulated activity</td><td>Li et al., 2016; Ishikawa-Sakurai et al., 2014</td></tr><tr><td>cold</td><td>nd<br>OsCPK17</td><td>AtPIP1;4 and AtPIP2;5 gene<br>expression<br>OsPIP2;1 and OsPIP2;6 C-ter<br>phosphorylation</td><td>up-regulation of gene<br>expression<br>up-regulated activity</td><td>Rahman et al., 2020<br>Almadanim et al., 2017</td></tr><tr><td>circadian<br>clock</td><td>14-3-3<br>ELF3</td><td>AtPIP2;1<br>AtPIP1;2 and AtPIP2;1 gene<br>expression</td><td>up-regulated activity<br>oscilating activity</td><td>Prado et al., 2019<br>Caldeira et al., 2014; Takase et al., 2011</td></tr><tr><td>flg22</td><td>BAK1 and<br>SnR2.6</td><td>increased AtPIP2;1 loop B<br>phosphorylation</td><td>up-regulated activity</td><td>Rodrigues et al., 2017</td></tr></tbody></table>
<table><tr><td rowspan="3">BIOTIC</td><td>Phytophthora sojae</td><td>CRN78</td><td>GmPIP2;13 and NbPIP2;2 phosphorylation</td><td>protein degradation</td><td>Ai et al., 2021</td></tr><tr><td>chitin</td><td>CERK1 and LYK5</td><td>nd</td><td>up-regulated activity</td><td>Attia et al., 2020</td></tr><tr><td>Xanthomonas oryzae</td><td>Harpin</td><td>interaction with OsPIP1;3 and AtPIP1;4</td><td>nd</td><td>Li et al., 2015b; Li et al., 2019</td></tr><tr><td rowspan="7">ENDOGENOUS</td><td>auxin</td><td>ARF7</td><td>PIP gene expression</td><td>down-regulated activity</td><td>Péret et al., 2012</td></tr><tr><td>MeJa</td><td>nd</td><td>PIP2 C-ter phosphorylation</td><td>up-regulated activity</td><td>Lee and Zwiazek, 2019</td></tr><tr><td rowspan="2">ethylene</td><td>RhETRs</td><td>RhPIP1;1 and RhPIP2;1 gene expression</td><td>down-regulated gene expression</td><td>Ma et al., 2008; Chen et al., 2013</td></tr><tr><td>ETR1 and CTR1</td><td>AtPIP2;1 phosphorylation</td><td>up-regulated activity</td><td>Qing et al., 2016; Yang et al., 2013</td></tr><tr><td rowspan="2">sucrose</td><td>SIRK1, QSK1</td><td>AtPIP2;6 phosphorylation</td><td>C-ter up-regulated activity</td><td>Niittyla et al., 2007; Wu et al., 2014; Wu et al., 2019; Wu et al., 2013</td></tr><tr><td>BSK8</td><td>AtPIP2;7 phosphorylation</td><td>C-ter nd</td><td>Wu et al., 2014</td></tr><tr><td>CIF peptide</td><td>SGN3</td><td>nd</td><td>down-regulated activity</td><td>Wang et al., 2019</td></tr><tr><td>Other</td><td>CASPL1B1</td><td>physical interaction with AtPIP2;1</td><td>up-regulated activity</td><td>Champeyroux et al., 2019</td></tr></table>
553 **Figure legends**

**Figure 1.** Cellular responses to biotic or abiotic stimuli involving hydrogen peroxide ($H_2O_2$) and aquaporins. **A**, In guard cells (right), the Snf1-Related protein Kinase 2.6 (SnRK2.6) is a key component of abscisic acid (ABA)- and flagellin (flg22)-induced stomatal closure. Flg22 is a Pathogen-Associated Molecular Pattern (PAMP) recognized by the plasma membrane Flagellin-Sensitive 2 (FLS2) receptor, which acts in concert with BRI1-Associated Kinase 1 (BAK1). Upon activation, SnRK2.6 phosphorylates two NADPH oxidases, Respiratory Burst Oxidase Homolog protein D (RBOHD) and RBOHF, and the AtPIP2;1 aquaporin. The latter facilitates the influx of $H_2O_2$ and the efflux of water, thereby promoting stomatal closure (Grondin et al. 2015; Rodrigues et al. 2017). Fgl22 also enhances the lateral mobility of AtPIP2;1 at the guard cell plasma membrane (left), thereby promoting its internalization (Cui et al. 2021). **B**, The root hydraulic conductivity ($Lp_r$) is strongly decreased in response to abiotic stresses such as osmotic and salt stress, or hormones such as salicylic acid (SA). All these stimuli act through accumulation of $H_2O_2$ which lowers AtPIP2;1 phosphorylation (P), alters its plasma membrane mobility and promotes its internalization (Boursiac et al. 2008; Wudick et al. 2015). In contrast, PIP phosphorylation promotes its trafficking to the plasma membrane. The increased apoplastic $H_2O_2$ production observed in response to an osmotic stress is mediated through the clustering in nanodomains of the Rho-related GTPase of plants 6 (Rop6) together with the RBOHD and F NADPH oxidases (Smokvarska et al. 2020; Martiniere et al. 2019). **C**, In mesophyll cells, PAMPs are perceived by plasma membrane receptors, which trigger, in combination with BAK1 co-receptor and other non-represented cellular components, the activation of RBOHB NADPH oxidase and, as a consequence, apoplastic accumulation of $H_2O_2$. The AtPIP1;4 aquaporin plays a key role in facilitating the diffusion of apoplastic $H_2O_2$ into the cytoplasm (Tian et al. 2016). The increase in cytoplasmic $H_2O_2$ concentration ultimately leads to activation of key proteins (Pathogenesis-Related protein 1: PR1; Pathogenesis-Related protein 2: PR2; Plant Defense 1.2: PDF1.2; Noninducer of PR genes 1: NPR1; Mitogen-Activated Protein Kinase 3: MAPK3; Glucan Synthase-like 5: GSL5) involved in Systemic Acquired Resistance (SAR) or Pathogen Triggered Immunity (PTI), as indicated. The figure also shows how the Crinkler 78 (CRN78) effector of *Phytophthora* can penetrate the plant cell where it interacts with phosphorylated NbPIP2;2, to induce its internalization and further degradation, thereby preventing its role in PAMP signaling (Ai et al., 2021). Thick blue arrows and thick red arrows indicate initial effects of stimuli and their output responses, respectively; thin dotted blue line arrows refer to diffusion of the indicated molecules; red dotted line arrows represent protein phosphorylation by indicated protein kinases; red dashed-dotted line arrows and red dashed-dotted line blunt arrows describe indirect activation and
586 inactivation pathways, respectively; brown line arrows indicate movements of proteins or membrane
587 compartments.

588

589 **Figure 2.** Signaling pathways converging on PIP phosphorylation. The figure represents a prototypal
590 PIP aquaporin with its transmembrane domains (1-6) and extracellular (A, C, E) or cytosolic (B, D)
591 loops. The indicated stimuli lead to enhanced (arrows) or reduced (blunt arrow) phosphorylation of
592 Ser residues in loop B or C-terminal tail. The inset shows three pathways acting on unknown PIP
593 phosphorylation sites (white question marks). Signaling intermediates are indicated with the
594 following color code: Snf1-related protein kinase 2 (SnRK2): blue; Calcium-Dependent Protein Kinase
595 (CDPK), green; Receptor-Like Kinase (RLK), red; others, black. Note that the indicated protein kinases
596 do not necessarily act directly on the indicated sites. Other abbreviations: Brassinosteroid Signaling
597 Kinase 8 (BSK8); Chitin Elicitor Receptor Kinase 1 (CERK1); Calcium-dependent Protein Kinase 17
598 (CPK17); Constitutive Triple-Response 1 (CTR1); Leaf Panicle 2 (LP2); Lysine motif receptor Kinase 5
599 (LYK5); phosphatidylinositol 3-kinase (PI3K); phosphatidylinositol 4-kinase (PI4K); Qian Shou Kinase 1
600 (QSK1); Receptor-like Kinase 1 (RKL1), Sucrose Induced Receptor Kinase 1 (SIRK1); 14-3-3 protein (14-
601 3-3). For more details and references see text and Table 1.

602

603
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929
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Cellular responses to biotic or abiotic stimuli involving hydrogen peroxide ($H_2O_2$) and aquaporins. **A**, In guard cells (right), the Snf1-Related protein Kinase 2.6 (SnRK2.6) is a key component of abscisic acid (ABA)- and flagellin (flg22)-induced stomatal closure. Flg22 is a Pathogen-Associated Molecular Pattern (PAMP) recognized by the plasma membrane Flagellin-Sensitive 2 (FLS2) receptor, which acts in concert with BRI1-Associated Kinase 1 (BAK1). Upon activation, SnRK2.6 phosphorylates two NADPH oxidases, Respiratory Burst Oxidase Homolog protein D (RBOHD) and RBOHF, and the AtPIP2;1 aquaporin. The latter facilitates the influx of $H_2O_2$ and the efflux of water, thereby promoting stomatal closure (Grondin et al. 2015; Rodrigues et al. 2017). Fgl22 also enhances the lateral mobility of AtPIP2;1 at the guard cell plasma membrane (left), thereby promoting its internalization (Cui et al. 2021). **B**, The root hydraulic conductivity ($L_p$) is strongly decreased in response to abiotic stresses such as osmotic and salt stress, or hormones such as salicylic acid (SA). All these stimuli act through accumulation of $H_2O_2$ which lowers AtPIP2;1 phosphorylation (P), alters its plasma membrane mobility and promotes its internalization (Boursiac et al. 2008; Wudick et al. 2015). In contrast, PIP phosphorylation promotes its trafficking to the plasma membrane. The increased apoplastic $H_2O_2$ production observed in response to an osmotic stress is mediated through the clustering in nanodomains of the Rho-related GTPase of plants 6 (Rop6) together with the RBOHD and F NADPH oxidases (Smokvarska et al. 2020; Martiniere et al. 2019). **C**, In mesophyll cells, PAMPs are perceived by plasma membrane receptors, which trigger, in combination with BAK1 co-receptor and other non-represented cellular components, the activation of RBOHB NADPH oxidase and, as a consequence, apoplastic accumulation of $H_2O_2$. The AtPIP1;4 aquaporin plays a key role in facilitating the diffusion of apoplastic $H_2O_2$ into the cytoplasm (Tian et al. 2016). The increase in cytoplasmic $H_2O_2$ concentration ultimately leads to activation of key proteins (Pathogenesis-Related protein PR1; Pathogenesis-Related protein 2: PR2; Plant Defensin 1.2: PDF1.2; Noninducer of PR genes 1: NPR1; Mitogen-activated Protein Kinase 3: MAPK3; Glucan Synthase-like 5: GSL5) involved in Systemic Acquired Resistance (SAR) or Pathogen Triggered Immunity (PTI), as indicated. The figure also shows how the Crinkler 78 (CRN78) effector of *Phytophthora* can penetrate the plant cell where it interacts with phosphorylated NbPIP2;2, to induce its internalization and further degradation, thereby preventing its role in PAMP signaling (Ai et al., 2021). Thick blue arrows and thick red arrows indicate initial effects of stimuli and their output responses, respectively; thin dotted blue line arrows refer to diffusion of the indicated molecules; red dotted line arrows represent protein phosphorylation by indicated protein kinases; red dashed-dotted line arrows and red dashed-dotted line blunt arrows describe indirect activation and inactivation pathways, respectively; brown line arrows indicate movements of proteins or membrane compartments.</figcaption></figure>
<figure><img src="image_4.png" /></figure>

**Figure 2.** Signaling pathways converging on PIP phosphorylation. The figure represents a prototypal PIP aquaporin with its transmembrane domains (1-6) and extracellular (A, C, E) or cytosolic (B, D) loops. The indicated stimuli lead to enhanced (arrows) or reduced (blunt arrow) phosphorylation of Ser residues in loop B or C-terminal tail. The inset shows three pathways acting on unknown PIP phosphorylation sites (white question marks). Signaling intermediates are indicated with the following color code: Snf1-related protein kinase 2 (SnRK2): blue; Calcium-Dependent Protein Kinase (CDPK), green; Receptor-Like Kinase (RLK), red; others, black. Note that the indicated protein kinases do not necessarily act directly on the indicated sites. Other abbreviations: Brassinosteroid Signaling Kinase 8 (BSK8); Chitin Elicitor Receptor Kinase 1 (CERK1); Calcium-dependent Protein Kinase 17 (CPK17); Constitutive Triple-Response 1 (CTR1); Leaf Panicle 2 (LP2); Lysine motif receptor Kinase 5 (LYK5); phosphatidylinositol 3-kinase (PI3K); phosphatidylinositol 4-kinase (PI4K); Qian Shou Kinase 1 (QSK1); Receptor-like Kinase 1 (RKL1), Sucrose Induced Receptor Kinase 1 (SIRK1); 14-3-3 protein (14-3-3). For more details and references see text and Table 1.
ADVANCES

* ABA-dependent and independent signalling pathways act on aquaporins in plants under water deficit.

* Aquaporin-mediated water transport is targeted by multiple abiotic, biotic, or endogenous stimuli; recently identified stimuli include peptide hormones and PAMPs.

* ROS play a central signalling role in regulating water transport in plants by acting on aquaporin subcellular dynamics, localization and/or phosphorylation.

* Plasma membrane aquaporins exhibit a dual water- and H₂O₂-transport function, acting as players in and targets of signalling processes.

* Phosphorylation of aquaporins, which interferes with their gating, trafficking, and stability, is a major target of plant signalling cascades, and multiple classes of protein kinases that act on distinct phosphorylation sites have been identified.
OUTSTANDING QUESTIONS

* Is aquaporin-mediated H₂O₂ transport a common process in plant cell signaling?

* Which cellular signals trigger RLK-dependent aquaporin regulation?

* What is the role of lipid signaling in water transport regulation?

* What are the mechanisms for coupling physical stimuli to aquaporin regulation?

* How do aquaporins contribute to plant defense? What are their active roles in plant immunity, and how can they be hijacked by pathogens?

* What are the effects of combined stresses on plant water transport, and which signaling pathways are involved?
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