2.1. PM H⁺-ATPases and pH-stats under tight control

PM H⁺-ATPases belong to the superfamily of P-type ATPases and consist of a single polypeptide chain folding into a multi-domain structure (Palmgren, Reference Palmgren2023). The core architecture of PM H⁺-ATPases follows the typical P-type ATPase structure, consisting of four domains: N (nucleotide-binding), P (phosphorylation), A (actuator) and M (membrane) domains that comprise 10 transmembrane domains (Kühlbrandt, Reference Kühlbrandt2004; Pedersen et al., Reference Pedersen, Buch-Pedersen, Preben Morth, Palmgren and Nissen2007). In yeast and plants, PM H⁺ ATPases carry sequence extensions at both their N- and C-termini, which are implicated in regulation and autoinhibition (Ekberg et al., Reference Ekberg, Palmgren, Veierskov and Buch-Pedersen2010; Falhof et al., Reference Falhof, Pedersen, Fuglsang and Palmgren2016; Kühlbrandt et al., Reference Kühlbrandt, Zeelen and Dietrich2002; Palmgren et al., Reference Palmgren, Sommarin, Serrano and Larsson1991). In the green lineage, PM H⁺-ATPases C-termini extend further into an R (regulatory) domain that includes multiple phosphorylation sites (Rudashevskaya et al., Reference Rudashevskaya, Ye, Jensen, Fuglsang and Palmgren2012). Cross-linking experiments showed that the R-domain can extensively cross-link with the A-, N- and P-domains, as well as the H⁺-binding site, suggesting that there is an intramolecular R-domain-mediated autoinhibition (Blackburn et al., Reference Blackburn, Nguyen, Patton, Bartosiak and Sussman2025; Nguyen et al., Reference Nguyen, Blackburn and Sussman2020).

PM H⁺-ATPases are normally autoinhibited at cytosolic pH but become strongly activated if the cytosol acidifies (Luo et al., Reference Luo, Morsomme and Boutry1999; Palmgren & Christensen, Reference Palmgren and Christensen1994; Regenberg et al., Reference Regenberg, Villalba, Lanfermeijer and Palmgren1995). The autoinhibition of the PM H⁺-ATPase can be overruled during signalling. Indeed, numerous signalling pathways in development, reproduction and immunity target the activity of the PM H⁺-ATPases and converge in modulating the phosphorylation level of the R-domain (Falhof et al., Reference Falhof, Pedersen, Fuglsang and Palmgren2016; Fuglsang & Palmgren, Reference Fuglsang and Palmgren2021; Haruta et al., Reference Haruta, Gray and Sussman2015). These studies unveiled a common regulatory scheme, an R-domain phosphocode, from which the contribution of individual residues, their hierarchy and the sequentiality of phosphorylation events emerge. The phosphorylation of the R-domain can be translated as positive or repressive marks, promoting or inhibiting PM H⁺-ATPase activity (Figure 1).

Of prominent importance is the phosphorylation of the conserved penultimate residue (a threonine; T947 in the Arabidopsis AUTOINHIBITED H⁺-ATPase 2; AHA2), which is commonly linked to AHA activation (Fuglsang et al., Reference Fuglsang, Visconti, Drumm, Jahn, Stensballe, Mattei, Jensen, Aducci and Palmgren1999; Svennelid et al., Reference Svennelid, Olsson, Piotrowski, Rosenquist, Ottman, Larsson, Oecking and Sommarin1999). Phosphorylation of this residue creates a binding site for 14-3-3 proteins, which, upon binding, releases C-terminal autoinhibition (Fuglsang et al., Reference Fuglsang, Visconti, Drumm, Jahn, Stensballe, Mattei, Jensen, Aducci and Palmgren1999; Jahn et al., Reference Jahn, Fuglsang, Olsson, Brüntrup, Collinge, Volkmann, Sommarin, Palmgren and Larsson1997). Another positive mark is the phosphorylation at T881 (AHA2) (Fuglsang et al., Reference Fuglsang, Kristensen, Cuin, Schulze, Persson, Thuesen, Ytting, Oehlenschlæger, Mahmood, Sondergaard, Shabala and Palmgren2014; Hayashi et al., Reference Hayashi, Fukatsu, Takahashi, Kinoshita, Kato, Sakakibara, Kuwata and Kinoshita2024; Wang et al., Reference Wang, Xie, Tan, Li, Wang, Pei, Li, Guo, Gong and Wang2022). Interestingly, time-resolved quantitative phosphoproteomics and genetic experiments indicate that in response to blue light, phosphorylation of the penultimate residue precedes and conditions the phosphorylation of T881 (pT881) in AHA1 (Hayashi et al., Reference Hayashi, Fukatsu, Takahashi, Kinoshita, Kato, Sakakibara, Kuwata and Kinoshita2024). This functional relationship appears unilateral as pT881 is dispensable for the phosphorylation of T947 (pT947) (Fuglsang et al., Reference Fuglsang, Kristensen, Cuin, Schulze, Persson, Thuesen, Ytting, Oehlenschlæger, Mahmood, Sondergaard, Shabala and Palmgren2014). In contrast to the pT947 and pT881, phosphorylation at S931 (pS931) (AHA2) corresponds to a repressive mark linked to AHA inhibition. pS931 prevents 14-3-3 binding to the R-domain independently of the phosphorylation status of T947 (Duby et al., Reference Duby, Poreba, Piotrowiak, Bobik, Derua, Waelkens and Boutry2009; Fuglsang et al., Reference Fuglsang, Guo, Cuin, Qiu, Song, Kristiansen, Bych, Schulz, Shabala, Schumaker, Palmgren and Zhub2007). Similarly, the phosphorylation of S899 (pS899) (AHA2) has been linked with inhibition of PM H⁺-ATPases (Haruta et al., Reference Haruta, Sabat, Stecker, Minkoff and Sussman2014; Zhu et al., Reference Zhu, Krall, Li, Xi, Luo, Li, He, Yang, Zan, Gilbert, Gombos, Wang, Neuhäuser, Jacquot, Lejay, Zhang, Liu, Schulze and Wu2024). How pS899 inhibits AHA activity, and its relationship with other residues, is, however, unknown. Similarly, the function of other residues identified in phosphoproteomic experiments remains unknown (Fuglsang & Palmgren, Reference Fuglsang and Palmgren2021; Rudashevskaya et al., Reference Rudashevskaya, Ye, Jensen, Fuglsang and Palmgren2012).

2.2. Molecular circuitry of auxin-mediated regulation of pHe and growth

The phytohormone auxin controls many processes in plants, including cell expansion, cell division and cell differentiation (Du et al., Reference Du, Spalding and Gray2020; Enders & Strader, Reference Enders and Strader2015). Auxin is perceived at the cell surface via the auxin-binding proteins (ABPs) and their plasma membrane partner, the transmembrane kinases (TMKs) (Friml et al., Reference Friml, Gallei, Gelová, Johnson, Mazur, Monzer, Rodriguez, Roosjen, Verstraeten, Živanović, Zou, Fiedler, Giannini, Grones, Hrtyan, Kaufmann, Kuhn, Narasimhan, Randuch and Rakusová2022), and intracellularly by TIR/AFB–Aux/IAA receptor–coreceptor modules (Dharmasiri et al., Reference Dharmasiri, Dharmasiri, Weijers, Lechner, Yamada, Hobbie, Ehrismann, Jürgens and Estelle2005; Kepinski & Leyser, Reference Kepinski and Leyser2005; Tan et al., Reference Tan, Calderon-Villalobos, Sharon, Zheng, Robinson, Estelle and Zheng2007). Auxin perception induces two main signalling branches: (i) a fast cellular auxin response that includes PM depolarization, changes in extracellular pH (pHe), Ca²⁺ influx and root growth inhibition (Ayling et al., Reference Ayling, Brownlee and Clarkson1994; Barbez et al., Reference Barbez, Dünser, Gaidora, Lendl and Busch2017; Monshausen et al., Reference Monshausen, Miller, Murphy and Gilroy2011); and (ii) a slower nuclear auxin pathway regulating a broad transcriptional response (Leyser, Reference Leyser2018; Weijers & Wagner, Reference Weijers and Wagner2016) (Figure 2). The effect of auxin on pHe is tissue-, concentration- and time-dependent.

Figure 2. Molecular circuitry of auxin-mediated regulation of extracellular pH. Auxin signalling is categorized into two main branches: fast non-transcriptional cellular responses and slower transcriptional responses that converge in the regulation of pHe. ABP1, AUXIN-BINDING PROTEIN 1; AFB, AUXIN-SIGNALLING F-BOX; AUX1, AUXIN-RESISTANT 1; FER, FERONIA; IAA, Indole-3-Acetic Acid; LLG1, LORELEI-LIKE GPI-ANCHOR PROTEIN 1; PP2C-D, type 2C protein phosphatase clade D; RALF1, RAPID ALKALINIZATION FACTOR1; SAUR19, SMALL AUXIN Up-RNA 19; TIR1, TRANSPORT INHIBITOR RESPONSE 1; TMK1, TRANSMEMBRANE KINASE 1. It is noteworthy that the stoichiometry of H⁺/IAA- cotransport by AUX1 is still a matter of debate (Geisler & Dreyer, Reference Geisler and Dreyer2024).

In Arabidopsis hypocotyls, transcriptional and non-transcriptional auxin responses converge towards the activation of PM H⁺-ATPases, the acidification of the apoplast and the promotion of cellular expansion (Fendrych et al., Reference Fendrych, Leung and Friml2016; Lin et al., Reference Lin, Zhou, Tang, Takahashi, Pan, Dai, Ren, Zhu, Pan, Zheng, Gray, Xu, Kinoshita and Yang2021; Ren et al., Reference Ren, Park, Spartz, Wong and Gray2018; Spartz et al., Reference Spartz, Ren, Park, Grandt, Lee, Murphy, Sussman, Overvoorde and Gray2014). Upon auxin perception by ABP1-TMK1, TMK1 phosphorylates and activates the PM H⁺-ATPase AHA1 within seconds (Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021; Lin et al., Reference Lin, Zhou, Tang, Takahashi, Pan, Dai, Ren, Zhu, Pan, Zheng, Gray, Xu, Kinoshita and Yang2021), providing a direct molecular link between auxin perception and acidification of the apoplast. Among the transcriptional targets regulated by auxin and its intracellular receptor is SMALL AUXIN Up-RNA 19 (SAUR19). SAUR19 binds to and inhibits the TYPE 2C PROTEIN PHOSPHATASES, which normally dephosphorylates and inhibits the activity of the PM H⁺-ATPases. Thereby, SAUR19 promotes PM H⁺-ATPase activity, acidification of the apoplast and cell expansion (Fendrych et al., Reference Fendrych, Leung and Friml2016; Ren et al., Reference Ren, Park, Spartz, Wong and Gray2018; Spartz et al., Reference Spartz, Ren, Park, Grandt, Lee, Murphy, Sussman, Overvoorde and Gray2014). Recently, pHe was proposed to be a key switch in auxin-induced hypocotyl elongation. Auxin-driven acidification promotes elongation until an optimal pHe threshold is reached (Wang et al., Reference Wang, Jin, Deng, Zheng, Guo, Ji, Song, Zeng, Kinoshita, Liao, Chen, Deng and Wei2025). Beyond this optimal pHe, elongation ceases due to a negative feedback loop, which results in a biphasic hypocotyl elongation and suggests a ‘gas then break’ mechanism for the fine-tuning of hypocotyl growth. Additional variables might be involved in this mechanism, such as light signalling, which antagonizes the influence of auxin on both pHe and cell elongation (Wang et al., Reference Wang, Jin, Deng, Zheng, Guo, Ji, Song, Zeng, Kinoshita, Liao, Chen, Deng and Wei2025). In Arabidopsis root, endogenous auxin signalling is required for apoplast acidification, cellular elongation (Barbez et al., Reference Barbez, Dünser, Gaidora, Lendl and Busch2017; Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021) and guide gravitropic and hydotropic root navigation in the soil environment.

2.3. Auxin-induced extracellular alkalinization in roots

Applied exogenously, nanomolar auxin concentrations trigger fast and reversible inhibition of root growth, which is linked to an alkalinization of the apoplast (Barbez et al., Reference Barbez, Dünser, Gaidora, Lendl and Busch2017; Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021). This suggests that auxin promotes an inward H⁺ flow, evoking alkalinization of the apoplast and accompanying depolarization of the PM. The auxin-triggered alkalinization relies on the IAA/H⁺ symporter AUX1, the non-transcriptional action of the intracellular auxin receptors and a CNGC14-mediated Ca²⁺ influx (Dindas et al., Reference Dindas, Scherzer, Roelfsema, Von Meyer, Müller, Al-Rasheid, Palme, Dietrich, Becker, Bennett and Hedrich2018; Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021; Serre et al., Reference Serre, Kralík, Yun, Slouka, Shabala and Fendrych2021, Reference Serre, Wernerová, Vittal, Dubey, Medvecká, Jelínková, Petrášek, Grossmann and Fendrych2023). AUX1 IAA/H⁺ symporter activity is expected to directly contribute to H⁺ influx. However, despite the fact that loss of AUX1 abolishes membrane depolarization in root hairs (Dindas et al., Reference Dindas, Scherzer, Roelfsema, Von Meyer, Müller, Al-Rasheid, Palme, Dietrich, Becker, Bennett and Hedrich2018), AUX1 has a minor contribution to auxin-induced membrane depolarization at the root tip (Serre et al., Reference Serre, Kralík, Yun, Slouka, Shabala and Fendrych2021) and the intracellular injection of auxin is sufficient to induce depolarization (Dindas et al., Reference Dindas, Scherzer, Roelfsema, Von Meyer, Müller, Al-Rasheid, Palme, Dietrich, Becker, Bennett and Hedrich2018). Furthermore, comparative analysis of the speed of H⁺ influx and auxin uptake indicates that auxin transport itself cannot fully explain the H⁺ influx, which would be predominantly executed by an unknown auxin-stimulated H⁺ permeable channel (Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021). Many signalling pathways crosstalk with auxin signalling and converge in the regulation of pHe and H⁺-ATPase. For instance, the cell surface receptor modules of both brassinosteroid and auxin directly phosphorylate and activate PM H⁺-ATPases to promote wall acidification and cell expansion (Miao et al., Reference Miao, Russinova and Rodriguez2022). Conversely, in the root, the perception of RAPID ALKALINIZATION FACTOR peptides (RALFs) and auxin converge in pHe alkalinization (Abarca et al., Reference Abarca, Franck and Zipfel2021; Gjetting et al., Reference Gjetting, Mahmood, Shabala, Kristensen, Shabala, Palmgren and Fuglsang2020; Li et al., Reference Li, Gallei and Friml2022; Morato do Canto et al., Reference Morato do Canto, Ceciliato, Ribeiro, Ortiz Morea, Franco Garcia, Silva-Filho and Moura2014). The effect of RALF1 on apoplastic pH is proposed to be linked to the inhibition of the PM H⁺-ATPase (Haruta et al., Reference Haruta, Sabat, Stecker, Minkoff and Sussman2014), albeit genetic experiments rather suggest that the activation of an unknown influx carrier is responsible for RALF1-triggered net H⁺ influx (Li et al., Reference Li, Gallei and Friml2022). Furthermore, RALF1 perception promotes auxin biosynthesis and signalling, thereby sustaining its effect on growth (Li et al., Reference Li, Gallei and Friml2022).

Figure 3. Plasma membrane H⁺-ATPases are central molecular nodes in immunity. Both pattern-triggered immunity and effector-triggered immunity converge on the regulation of PM H⁺-ATPases. Pathogens evolved an array of strategies to inhibit or activate PM H⁺-ATPases. The plant molecular components are named in green, and pathogen-derived molecules are named in red. CNLs, coil-coiled nucleotide-binding leucine-rich repeat receptors; FER, FERONIA; LLG1, LORELEI-LIKE GPI-ANCHOR PROTEIN 1; PRR, pattern recognition receptor; RALF, RAPID ALKALINIZATION FACTOR; RIN4, RPM1-INTERACTING PROTEIN4.

As in the hypocotyl, TMK1-mediated activation of PM H⁺-ATPases occurs in response to auxin treatment and counterbalances the dominating alkalinization (Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021). This intuitively argues against PM H⁺-ATPases playing a role in the auxin-induced alkalinization, as it is explained by a net H⁺ influx, the mechanism of which remains unknown (Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021, Reference Li, Gallei and Friml2022; Serre et al., Reference Serre, Wernerová, Vittal, Dubey, Medvecká, Jelínková, Petrášek, Grossmann and Fendrych2023). However, the PM H⁺-ATPase can also attain an uncoupled state that is either leaky to H⁺ or exhibits slippage (ATP hydrolysis without accompanying H⁺ transport) (Baunsgaard et al., Reference Baunsgaard, Venema, Axelsen, Villalba, Welling, Wollenweber and Palmgren1996; Morsomme et al., Reference Morsomme, De Kerchove D’Exaerde, De Meester, Thinès, Goffeau and Boutry1996; Pedersen et al., Reference Pedersen, Kanashova, Dittmar and Palmgren2018). Similarly, the animal Na⁺/K⁺-ATPase, a related P-type ATPase, can be converted into a channel protein that causes the direction of Na⁺ transport to be downhill (Reyes & Gadsby, Reference Reyes and Gadsby2006; Scheiner-Bobis & Schneider, Reference Scheiner-Bobis and Schneider1997). Whether the uncoupling of the PM H⁺-ATPase contributes to the initial rapid extracellular alkalinization in roots remains to be investigated.

2.4. PM H⁺-ATPases as convergent nodes linking pattern- and effector-triggered immunities

Changes in pHe have been repetitively observed in plant–microbe interactions (Elmore & Coaker, Reference Elmore and Coaker2011; Felle et al., Reference Felle, Herrmann, Hanstein, Hückelhoven and Kogel2004; Kesten et al., Reference Kesten, Gámez-Arjona, Menna, Scholl, Dora, Huerta, Huang, Tintor, Kinoshita, Rep, Krebs, Schumacher and Sánchez‐Rodríguez2019; Vera-Estrella et al., Reference Vera-Estrella, Barkla, Higgins and Blumwald1994). Plants perceive microbes via cell surface and intracellular immune receptors involved in pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) (Ngou et al., Reference Ngou, Ding and Jones2022). At the cell surface, pattern recognition receptors (PPRs) sense microbe- or self-derived molecular patterns and initiate an array of molecular events culminating in PTI (DeFalco & Zipfel, Reference DeFalco and Zipfel2021). Together with the production of reactive oxygen species and influx of Ca²⁺, the rapid alkalinization of the apoplast corresponds to a rapid molecular hallmark of PPR signalling (Boller & Felix, Reference Boller and Felix2009) (Figure 3). Indeed, rapid changes in apoplastic pH have been observed in various plant species irrespective of the biochemical nature and the microbial origin of the molecular pattern perceived. For instance, perception of the fungal cell wall component chitin, the oomycete-derived peptide pep-13, the bacterial flagellin epitope flg22 and the endogenous danger-associated molecular patterns pep1 all lead to alkalinization of pHe (Felix et al., Reference Felix, Regenass and Boller1993; Liu et al., Reference Liu, Song, Huang, Jiang, Moriwaki, Wang, Men, Zhang, Wen, Han, Chai and Guo2022; Nürnberger et al., Reference Nürnberger, Nennstiel, Jabs, Sacks, Hahlbrock and Scheel1994; Yamaguchi et al., Reference Yamaguchi, Pearce and Ryan2006). Untargeted phosphoproteomic studies indicate that it may be mediated by the inhibition of PM H⁺ ATPases, as the perception of flg22 leads to a decrease in the phosphorylation of residues known to promote PM H⁺-ATPase activity (e.g., AHA2 T947; Benschop et al., Reference Benschop, Mohammed, O’Flaherty, Heck, Slijper and Menke2007; Nühse et al., Reference Nühse, Bottrill, Jones and Peck2007), and the perception of flg22 and oligogalacturonides promotes the phosphorylation of a residue linked to the inhibition of the PM H⁺-ATPase (AHA2 S899; Benschop et al., Reference Benschop, Mohammed, O’Flaherty, Heck, Slijper and Menke2007; Mattei et al., Reference Mattei, Spinelli, Pontiggia and De Lorenzo2016; Nühse et al., Reference Nühse, Bottrill, Jones and Peck2007).

Akin to their cell surface counterpart, intercellular immune receptors have been shown to modulate pHe. Indeed, in Nicotiana benthamiana several members of a subgroup of intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), namely the plasma membrane-localized coiled-coil NLRs (CNLs) (Saile et al., Reference Saile, Ackermann, Sunil, Keicher, Bayless, Bonardi, Wan, Doumane, Stöbbe, Jaillais, Caillaud, Dangl, Nishimura, Oecking and El Kasmi2021), have been reported to inhibit PM H⁺ ATPase activity, resulting in apoplastic alkalization (Lee et al., Reference Lee, Seo, Lee, Lee, Oh, Kim, Jung, Kim, Park, Kim, Mang and Choi2022). Interestingly, genetic and pharmacologic approaches indicate that inhibition and hyper-activation of PM H⁺ ATPase activity affect CNL function (Lee et al., Reference Lee, Seo, Lee, Lee, Oh, Kim, Jung, Kim, Park, Kim, Mang and Choi2022). Upon activation, CNLs form a PM-localized Ca²⁺-permeable wheel-like oligomeric structures mediating a Ca²⁺ influx for ETI signalling (Wang et al., Reference Wang, Song and Chai2023). Whether balanced H⁺-ATPase activity is required for the activation of CNLs or CNL-mediated execution of cell death is currently unknown. The two CNLs RPS2 and RPM1 guard the plant protein RPM1-INTERACTING PROTEIN4 (RIN4) and activate ETI upon sensing effector-mediated RIN4 modification (Axtell & Staskawicz, Reference Axtell and Staskawicz2003; Mackey et al., Reference Mackey, Holt, Wiig and Dangl2002). Interestingly, RIN4 was shown to directly associate and promote the activity of H⁺ ATPase (Liu et al., Reference Liu, Elmore, Fuglsang, Palmgren, Staskawicz and Coaker2009). Further, during infection, in the presence of the Pseudomonas syringae effector AvrB, RPM1-INDUCED PROTEIN KINASE phosphorylates RIN4 at T166, thereby promoting its association with AHA1 (Lee et al., Reference Lee, Bourdais, Yu, Robatzek and Coaker2015). As flg22 perception inhibits RIN4 phosphorylation at this residue, the inhibition of PM H⁺-ATPase upon flg22-triggered signalling may, in part, be mediated by the regulation of RIN4 (Lee et al., Reference Lee, Bourdais, Yu, Robatzek and Coaker2015). Altogether, these studies place PM H⁺-ATPases as a central node linking the two main branches of the plant immune system, which may contribute to their functional relationship (Feehan et al., Reference Feehan, Wang, Sun, Choi, Ahn, Ngou, Parker and Jones2023; Ngou et al., Reference Ngou, Ahn, Ding and Jones2021; Wang et al., Reference Wang, Song and Chai2023).

Interestingly, PM H⁺-ATPases are among the limited number of proteins differentially phosphorylated upon both ETI and PTI (Kadota et al., Reference Kadota, Liebrand, Goto, Sklenar, Derbyshire, Menke, Torres, Molina, Zipfel, Coaker and Shirasu2019). Given the apparent importance of PM H⁺-ATPase in immune signalling, it is not surprising that it is a common target of pathogens that promote disease. Indeed, pathogens have been shown to utilize metabolites and protein effectors to manipulate PM H⁺-ATPase (Figure 3). A prominent example is fusicoccin, a diterpene glucoside produced by the pathogenic fungus Fusicoccum amygdale, which irreversibly stabilizes the interaction between the 14-3-3 and the C-terminal regulatory domain of PM H⁺-ATPases (Baunsgaard et al., Reference Baunsgaard, Fuglsang, Jahn, Korthout, De Boer and Palmgren1998; Jahn et al., Reference Jahn, Fuglsang, Olsson, Brüntrup, Collinge, Volkmann, Sommarin, Palmgren and Larsson1997), thereby relieving auto-inhibition and leading to constitutive H⁺ pumping. As stomatal pores open in response to activation of H⁺ pumping by the PM H⁺-ATPase (Cha et al., Reference Cha, Min and Seo2024; Hayashi et al., Reference Hayashi, Fukatsu, Takahashi, Kinoshita, Kato, Sakakibara, Kuwata and Kinoshita2024; Kinoshita & Shimazaki, Reference Kinoshita and Shimazaki1999), this provides an entry point for the pathogen. Fusicoccin has been shown to cause continuous stomatal opening, wilting and necrosis of leaves (Elmore & Coaker, Reference Elmore and Coaker2011). By contrast, tenuazonic acid produced by fungi, such as Stemphylium loti, inhibits PM H⁺-ATPase activity (Bjørk et al., Reference Bjørk, Rasmussen, Gjetting, Havshøi, Petersen, Ipsen, Larsen and Fuglsang2020; Havshøi et al., Reference Havshøi, Nielsen and Fuglsang2024). Several protein effectors have been shown to target PM H⁺-ATPase activity as well. For instance, the Phytophthora infestans RxLR effector PITG06478 hijacks 14-3-3 proteins to suppress PM H⁺-ATPase activity (Seo et al., Reference Seo, Yan, Choi and Mang2023), and the Phytophthora capsici effector CRISIS2 associates with and inhibits PM H⁺-ATPases to promote disease (Seo et al., Reference Seo, Yan, Choi and Mang2023). By disrupting PM H⁺-ATPase function, these effectors are proposed to facilitate successful microbial proliferation. However, the underlying molecular mechanisms remain unknown. In addition, pathogens and parasites utilize plant mimicking RALF peptides to subvert PM H⁺-ATPases to their advantage (Masachis et al., Reference Masachis, Segorbe, Turrà, Leon-Ruiz, Fürst, El Ghalid, Leonard, López-Berges, Richards, Felix and Di Pietro2016; Wang et al., Reference Wang, Liu, Yuan, Chen, Zhao, Ali, Zheng, Tan, Yao, Zheng, Wu, Xu, Shi, Wu, Gao and Gu2024; Wood et al., Reference Wood, Walker, Lee, Urban and Hammond-Kosack2020; Zhang et al., Reference Zhang, Peng, Zhu, Xing, Li, Zhu, Zheng, Wang, Wang, Chen, Ming, Yao, Jian, Luan, Coleman-Derr, Liao, Peng, Peng and Yu2020). Still, many key questions remain to be answered. For instance, how signalling is relayed from activated immune complexes to the PM H⁺-ATPase is currently unknown. Further, while the regulation of PM H⁺-ATPase has been functionally linked with the regulation of stomatal opening, thereby limiting the entry of bacteria inside plant tissues (Liu et al., Reference Liu, Elmore, Fuglsang, Palmgren, Staskawicz and Coaker2009; Melotto et al., Reference Melotto, Underwood, Koczan, Nomura and He2006), the role of pHe in immune signalling and plant immunity in other cell types remains unclear.

2.5. Feedback loops in pH sensing and signalling

How the plant cell senses pHe has long been enigmatic (Tsai & Schmidt, Reference Tsai and Schmidt2021). Accumulating evidence from structural biology studies emphasizes the implication of cell surface ligand–receptor modules in pH sensing and signalling (Xu & Yu, Reference Xu and Yu2023). For instance, the brassinosteroid-induced association between the main brassinosteroid receptor Brassinosteroid-Insensitive1 (BRI1) and its co-receptor BRI1-Associated Receptor Kinase1 (BAK1) is promoted by a relatively acidic pH in vitro (Sun et al., Reference Sun, Han, Tang, Hu, Chai, Zhou and Chai2013). On the contrary, relatively alkaline pH promotes the binding of the peptide pep1 to its corresponding receptors PEPR1 and PEPR2 (Liu et al., Reference Liu, Song, Huang, Jiang, Moriwaki, Wang, Men, Zhang, Wen, Han, Chai and Guo2022; Tang et al., Reference Tang, Han, Sun, Zhang, Gong and Chai2015). This is explained by reversible (de-)protonation of amino acid residues and such direct interaction with H⁺, which occurs within the ligand or at the interface between the receptor and co-receptors (Xu & Yu, Reference Xu and Yu2023). These observations recently found a biological echo in the balance between plant immunity and growth. Indeed, pep1-triggered immune signalling alkalinizes pHe in the root apical meristem, thereby inhibiting the perception of root meristem growth factor 1 by its corresponding receptor, and ultimately growth (Liu et al., Reference Liu, Song, Huang, Jiang, Moriwaki, Wang, Men, Zhang, Wen, Han, Chai and Guo2022).

Several of these cell surface receptors have been shown to associate and directly phosphorylate AHAs. For instance, as discussed above, auxin perception induces TMK1–AHA1 interaction and AHA1 phosphorylation by TMK1 (Li et al., Reference Li, Verstraeten, Roosjen, Takahashi, Rodriguez, Merrin, Chen, Shabala, Smet, Ren, Vanneste, Shabala, De Rybel, Weijers, Kinoshita, Gray and Friml2021). Similarly, AHA1 associates with and is activated by BRI1 (Caesar et al., Reference Caesar, Elgass, Chen, Huppenberger, Witthöft, Schleifenbaum, Blatt, Oecking and Harter2011). Conversely, the receptor kinase Qiān Shŏu Kinase1 has been proposed to directly phosphorylate and inhibit AHA2 to mediate a low nitrate response (Zhu et al., Reference Zhu, Krall, Li, Xi, Luo, Li, He, Yang, Zan, Gilbert, Gombos, Wang, Neuhäuser, Jacquot, Lejay, Zhang, Liu, Schulze and Wu2024). How these stimuli-dependent associations are regulated is currently unknown. Finally, it is interesting to note that cell surface signalling pathways that are activated at relatively low pHe have been shown to promote AHA activity (Großeholz et al., Reference Großeholz, Wanke, Rohr, Glöckner, Rausch, Scholl, Scacchi, Spazierer, Shabala, Shabala, Schumacher, Kummer and Harter2022; Sun et al., Reference Sun, Han, Tang, Hu, Chai, Zhou and Chai2013). Conversely, cell surface pathways activated at relatively high pHe have been shown to inhibit AHA activity (Liu et al., Reference Liu, Song, Huang, Jiang, Moriwaki, Wang, Men, Zhang, Wen, Han, Chai and Guo2022; Tang et al., Reference Tang, Han, Sun, Zhang, Gong and Chai2015) (Figure 4). This suggests that cell surface pathways operate positive feed-forward loops to optimize selfishly their own signalling to the detriment of others. How this is balanced and regulated as part of the plant developmental programme is unknown.

Figure 4. Feedback loops in pH sensing and signalling. Through pH-sensitive ligand–receptor and ligand-induced receptor interactions, cell surface receptors and their corresponding ligands play preponderant role in pHe sensing. They can be categorized into two classes having relatively low or high pH optimum and signalling back to the PM H⁺-ATPases through feedback loops.

3.1. PM H⁺-ATPase oligomerization, strength in numbers?

Isolated monomers of PM H⁺-ATPase lacking the C-terminal autoinhibitory domain are functional in vitro in membrane nanodiscs (Justesen et al., Reference Justesen, Hansen, Martens, Theorin, Palmgren, Martinez, Pomorski and Fuglsang2013), suggesting that there is no strict additional molecular requirement for the H⁺ translocation capability. In plants, however, PM H⁺-ATPases exist in various oligomerization states (Kanczewska et al., Reference Kanczewska, Marco, Vandermeeren, Maudoux, Rigaud and Boutry2005). Consistent with higher-order assemblies of PM H⁺-ATPase, cryogenic electron microscopic (cryo-EM) studies of plant and fungal PM H⁺-ATPases resolved the organization and structure of hexamers (Cyrklaff et al., Reference Cyrklaff, Auer, Kühlbrandt and Scarborough1995; Heit et al., Reference Heit, Geurts, Murphy, Corey, Mills, Kühlbrandt and Bublitz2021; Ottmann et al., Reference Ottmann, Marco, Jaspert, Marcon, Schauer, Weyand, Vandermeeren, Duby, Boutry, Wittinghofer, Rigaud and Oecking2007; Zhao et al., Reference Zhao, Zhao, Chen, Yun, Li and Bai2021). In yeast, PM H⁺-ATPases appear to be produced and transported from the endoplasmic reticulum as hexamers (Lee et al., Reference Lee, Hamamoto and Schekman2002). In plants, PM H⁺-ATPase seems to be predominantly found in the form of dimers (Kanczewska et al., Reference Kanczewska, Marco, Vandermeeren, Maudoux, Rigaud and Boutry2005). The strong and irreversible activation induced by fusicoccin treatment leads to the formation of higher-order oligomers (Kanczewska et al., Reference Kanczewska, Marco, Vandermeeren, Maudoux, Rigaud and Boutry2005) and phosphorylation-dependent binding of 14-3-3 dimers to dimeric H⁺-ATPases is proposed to lead to the assembly of H⁺-ATPase hexamers (Ottmann et al., Reference Ottmann, Marco, Jaspert, Marcon, Schauer, Weyand, Vandermeeren, Duby, Boutry, Wittinghofer, Rigaud and Oecking2007). Cross-linking experiments further suggest ‘head-to-tail’ interactions between the R-domains of neighbouring monomers (Nguyen et al., Reference Nguyen, Blackburn and Sussman2020). However, whether PM H⁺-ATPase oligomerization occurs during plant signalling and what could be the associated functional consequences remain largely unclear. It is tempting to speculate that the formation of such oligomers could drive important and localized changes in pHe (Figure 5).

Figure 5. Unknown molecular assembly of plant plasma membrane H⁺-ATPases and surrounding lipids. Top view of schematic representation of the PM H⁺-ATPase transmembrane alpha helices. While the cryo-EM studies of plant, yeast and fungus highlight PM H⁺-ATPase homo-hexamers, the potential dynamic assembly of oligomers in vivo and their potential function remain largely unknown in plants. The light pink membrane areas depict a putative functional paralipidome in which specific lipids are hypothesized to fine-tune PM H⁺-ATPase activity. Bottom: Side view of PM H⁺-ATPase monomer and hexamer representing a putative phosphatidylserine-driven H⁺ funnelling and the generation of a pHe nano-environment.

PM H⁺-ATPases form a multigenic protein family in plants, with 11 isoforms encoded in the genome of Arabidopsis thaliana Col-0, for instance (Palmgren, Reference Palmgren2001). Whether the assembly of dimers and higher-order oligomers involves heteromerization of AHA isoforms is currently unknown. Co-immunoprecipitation experiments indicate that different isoforms can be found in proximity (Rodrigues et al., Reference Rodrigues, Sabat, Minkoff, Burch, Nguyen and Sussman2014) and suggest the formation of heteromeric complexes. Given the differences in H⁺ translocation efficiency and pH sensitivity among AHA isoforms (Hoffmann et al., Reference Hoffmann, Olsen, Ezike, Pedersen, Manstretta, López-Marqués and Palmgren2019; Reference Hoffmann, Portes, Olsen, Damineli, Hayashi, Nunes, Pedersen, Lima, Campos, Feijó and Palmgren2020), the potential formation of hetero-oligomers may serve for finely tuned pHe variation in cell-type and stimuli-dependent conditions.

3.2. A functional H⁺-ATPase paralipidome?

The sensitivity of H⁺-ATPases to lipid moieties has long been described (Palmgren et al., Reference Palmgren, Sommarin, Ulvskov and Jørgensen1988). For instance, phospholipid species, such as lyso-phosphatidylcholine (lyso-PC) (Palmgren & Sommarin, Reference Palmgren and Sommarin1989) and phosphatidylserine (PS) (Paweletz et al., Reference Paweletz, Holtbrügge, Löb, De Vecchis, Schäfer, Günther Pomorski and Justesen2023), have been shown to stimulate H⁺ pumping by the PM H⁺-ATPase. Membrane lipids can serve simultaneously as solvents and regulatory co-factors for membrane proteins, constituting a so-called paralipidome (Levental & Lyman, Reference Levental and Lyman2022). Further, PM lipids and proteins are dynamically organized into numerous membrane nano-environments termed nanodomains (Jaillais et al., Reference Jaillais, Bayer, Bergmann, Botella, Boutté, Bozkurt, Caillaud, Germain, Grossmann, Heilmann, Hemsley, Kirchhelle, Martinière, Miao, Mongrand, Müller, Noack, Oda, Ott and Gronnier2024). In the yeast Saccharomyces cerevisiae, the PM H⁺-ATPase1 (PMA1) is confined to subregions of the PM forming a reticulated mesh-like network that is spatially distinct from the membrane compartments formed by the amino acid transporter Canavanine-resistance1 (Can1) (Malínská et al., Reference Malínská, Malínský, Opekarová and Tanner2003; Spira et al., Reference Spira, Mueller, Beck, Von Olshausen, Beig and Wedlich-Söldner2012). A comparative lipidomic analysis of PMA1- and Can1-containing membrane nano-environments indicates that sphingolipids and PS are enriched in the vicinity of PMA1 (van’t Klooster et al., Reference van ’t Klooster, Cheng, Sikkema, Jeucken, Moody and Poolman2020).

Interestingly, very long chain-containing sphingolipids are required for the oligomerization and PM localization of yeast PMA1 (Lee et al., Reference Lee, Hamamoto and Schekman2002; Gaigg et al., Reference Gaigg, Toulmay and Schneiter2006; Wang & Chang, Reference Wang and Chang2002) and cryo-EM analysis showed that the PMA1 hexamer encircles lipids of the outer leaflet, forming a liquid-crystalline patch of membrane that is presumably composed of sphingolipids (Zhao et al., Reference Zhao, Zhao, Chen, Yun, Li and Bai2021). Plant and yeast PM H⁺-ATPases co-purify with detergent-resistant membrane biochemical fractions (Bagnat et al., Reference Bagnat, Chang and Simons2001; Mongrand et al., Reference Mongrand, Morel, Laroche, Claverol, Carde, Hartmann, Bonneu, Simon-Plas, Lessire and Bessoule2004) indicating that they present similar membrane biochemical properties, which may be linked to sphingolipids. Plant sphingolipids play preponderant roles in regulating PM structure and function (Gronnier et al., Reference Gronnier, Germain, Gouguet, Cacas and Mongrand2016; Mamode Cassim et al., Reference Mamode Cassim, Gouguet, Gronnier, Laurent, Germain, Grison, Boutté, Gerbeau-Pissot, Simon-Plas and Mongrand2019). Interestingly, the inhibition of ceramide synthase involved in sphingolipid synthesis by the fungal toxin fumonisin B1 (Wang et al., Reference Wang, Norred, Bacon, Riley and Merrill1991; Zhang et al., Reference Zhang, Fang, Xie and Gong2024) affects PM H⁺-ATPase activity in maize embryos (Gutiérrez-Nájera et al., Reference Gutiérrez-Nájera, Muñoz-Clares, Palacios-Bahena, Ramírez, Sánchez-Nieto, Plasencia and Gavilanes-Ruíz2005; Gutiérrez-Nájera et al., Reference Gutiérrez-Nájera, Saucedo-García, Noyola-Martínez, Vázquez-Vázquez, Palacios-Bahena, Carmona-Salazar, Plasencia, El-Hafidi and Gavilanes-Ruiz2020), suggesting that a functional interplay between PM H⁺-ATPases and sphingolipids exists in plants as well (Figures 4 and 5).

Molecular dynamic simulations of Neurospora crassa PMA1 oligomers predict that PS binds to PMA1 and accumulates at the interface between monomers (Heit et al., Reference Heit, Geurts, Murphy, Corey, Mills, Kühlbrandt and Bublitz2021). Similarly, molecular dynamic simulations of AHA2 indicate preferential association with PS (Paweletz et al., Reference Paweletz, Holtbrügge, Löb, De Vecchis, Schäfer, Günther Pomorski and Justesen2023). PS has been suggested to promote the assembly of PM H⁺-ATPase hexamers (Heit et al., Reference Heit, Geurts, Murphy, Corey, Mills, Kühlbrandt and Bublitz2021). Further, the polar head of PS being electronegative, its accumulation within the vicinity of PMA1 hexamer may serve to attract and funnel H⁺ to foster H⁺ translocation by the PM H⁺-ATPases (Heit et al., Reference Heit, Geurts, Murphy, Corey, Mills, Kühlbrandt and Bublitz2021) (Figure 5). Corroborating these predictions, PS is particularly efficient in promoting the activity of Arabidopsis AHA2 in proteoliposomes (Paweletz et al., Reference Paweletz, Holtbrügge, Löb, De Vecchis, Schäfer, Günther Pomorski and Justesen2023). However, the exact molecular mechanism for the regulation of H⁺-ATPases activity by PS remains to be determined. In comparison with yeast PMA1, which has served as a model protein to study PM organization (Malinsky et al., Reference Malinsky, Opekarová, Grossmann and Tanner2013), much less is known about the nanoscale organization of its plant homologues. In Arabidopsis, single-particle tracking photoactivated localization microscopy experiments showed that AHA2 diffuses slowly within the PM, and that osmotic stress enhances AHA2 diffusion (Martinière et al., Reference Martinière, Fiche, Smokvarska, Mari, Alcon, Dumont, Hematy, Jaillais, Nollmann and Maurel2019). Long-term single-molecule imaging indicates that slow diffusing AHA2 is transiently spatially confined in membrane nano-environments (von Arx et al., Reference von Arx, Xhelilaj, Schulz, Oven-Krockhaus and Gronnier2024). The molecular bases of these events and their functional relevance are, however, unknown. It would be of particular interest to investigate the potential dynamic formation of PM H⁺-ATPase complexes and potential nanoclusters in living plant cells.