Plasma membrane (PM) H+-ATPases belong to the P-type subfamily of H+-ATPases and are located at the plasma membrane where they export cytoplasmic protons into the apoplast, generating proton gradients and electrical potential differences (Palmgren, 2001; Falhof et al., 2016). Each PM H+-ATPase consists of a single protein that is encoded by a multigene family in plants. The Arabidopsis (Arabidopsis thaliana) genome, for instance, includes 11 members, named Arabidopsis thaliana PLASMA MEMBRANE PROTON ATPASE1 (AHA1, also meaning Autoinhibited H+-ATPase isoform 1) to AHA11 (Palmgren, 2001). PM H+-ATPases have also been identified in Leadwort-leaved tobacco (Nicotiana plumbaginifolia), with nine members named PLASMA MEMBRANE PROTON ATPASE1 (PMA1) to PMA9, and in Oryza sativa, with 10 members named Oryza sativa H+-ATPase1 (OSA1) to OSA10 (Oufattole et al., 2000; Arango et al., 2003). These PM H+-ATPase isoforms exhibit high identity (Palmgren, 1998).

In Arabidopsis, AHA2 contains 10 transmembrane domains and 5 cytosolic domains: the N terminus, the actuator (A) domain, the nucleotide (N) binding domain, the phosphorylation (P) domain, and the regulation (R) domain (Palmgren, 2001; Morth et al., 2010). The A, N, and P domains play roles in catalytic activity, while the N terminus and R domain are responsible for activity regulation. Indeed, the R domain is a C-terminal autoinhibitory domain that contains two autoinhibitory regions, R1 and R2, which has been demonstrated to be important for the regulation of H+-ATPase activity, as its removal in AHA2 leads to a constitutively active H+-ATPase (Palmgren, 1998). Experimental evidence also showed that the N terminus controls the pump activity (Ekberg et al., 2010). A PM H+-ATPase exists in two major enzymatic states, E1 and E2. The E1 form displays a high affinity for ATP and H+ and exports cytoplasmic protons to the apoplast, thus generating a proton gradient via ATP hydrolysis. The E2 form has a much lower affinity for ATP and H+ but exhibits a high affinity for the inhibitor sodium orthovanadate (Na3VO4). The E1 form converts to the E2 form through phosphorylation of a conserved Asp residue within the DKTGT motif in the P domain (Asp329 in AHA2), whereby the H+ is released to apoplast due to the low H+ affinity of the E2 form. The E2 form then reverts to E1 upon dephosphorylation of the P domain by hydrolysis of the Asp-phosphate bond (Palmgren, 2001; Morth et al., 2010).

Due to its central role in plant physiological processes, PM H+-ATPase activity is finely regulated at the transcriptional and posttranslational levels. Phosphorylation or dephosphorylation of PM H+-ATPase is a major posttranslational modification. Six phosphorylation sites in the C-terminal domain (Thr-881, Ser-899, Thr-924, Ser-931, Tyr-946, and Thr-947 in Arabidopsis AHA2) are identified and their function is investigated. Phosphorylation of Thr-947 creates a binding motif, 946YpTV, for interaction with the 14-3-3 class of chaperones. Binding of 14-3-3 leads to AHA2 activation by alleviating autoinhibition (Fuglsang et al., 1999; Fuglsang et al., 2007). Phosphorylation of Thr-924 and Tyr-946 has also been reported to promote binding of 14-3-3 to the 946YpTV motif, thus activating AHA2 (Fuglsang et al., 1999; Fuglsang et al., 2003). The PROTEIN KINASE SOS2-LIKE5 (PKS5) phosphorylates AHA2 at Ser-931 and blocks the interaction between AHA2 and 14-3-3 (Fuglsang et al., 2007), leading to the inhibition of AHA2 activity. By contrast, the protein kinase PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 RECEPTOR (PSY1R) interacts with and phosphorylates AHA2/AHA1 at Thr-881 to activate the pumps (Fuglsang et al., 2014). RAPID ALKALIZATION FACTOR (RALF) peptides are sensed by the kinase FERONIA, which then phosphorylates Ser-899 of AHA2 to inhibit proton transport (Haruta et al., 2014). In addition, several additional phosphorylation sites were detected through tandem mass spectrometry, but their functions remain unknown (Falhof et al., 2016). The dephosphorylation of key phosphosites in PM H+-ATPase usually have effects opposite to those caused by phosphorylation. D clade type 2C protein phosphatases (PP2Cs) have been reported to negatively regulate PM H+-ATPase activity by interacting with AHA2 and dephosphorylate the Thr-947 site (Ren et al., 2018). Recently, the clade A PP2C ABSCISIC ACID INSENSITIVE1 (ABI1) was shown to interact with the AHA2 C terminus and dephosphorylate the Thr-947 residue to decrease pump activity (Miao et al., 2021).

Plants constantly cope with various abiotic stresses from their surrounding environment, such as salt and high pH, temperature, drought, light stress, macronutrient deficiency, acidic soil and aluminum stress, as well as heavy metal toxicity. PM H+-ATPase plays important roles in the responses to these adverse abiotic conditions. Here, we review recent discoveries of PM H+-ATPases involved in abiotic stress responses and provide insight into the mechanism by which plants evoke tolerance to abiotic stress, as well as a theoretical basis for improving plant stress tolerance by genetic engineering.

PM H+-ATPases, known as master enzymes, play significant roles in plant growth, development, and abiotic stress responses (Havshøi and Fuglsang, 2022). PM H+-ATPases participate in many important physiological processes, such as pH homeostasis, stomatal opening, nutrient uptake, phloem loading, and hypocotyl cell elongation (Palmgren, 2001; Chen et al., 2020a; Ding et al., 2021).

PM H+-ATPases regulate intracellular and extracellular pH homeostasis, which directs plant growth. Indeed, the classical acid-growth theory postulates that auxin-induced cell wall loosening initiates cell expansion, with PM H+-ATPases being essential (Rayle and Cleland, 1970). PM H+-ATPases excrete protons from the cytoplasm to the apoplast, leading to apoplastic acidification whose resulting lower acidic pH (3.5–4.0) is necessary for cell wall loosening (Palmgren, 1998). Auxin was reported to activate PM H+-ATPases and achieve apoplastic acidification (Moloney et al., 1981). This auxin-induced acidification and growth involve auxin perception in the nucleus via the TRANSPORT INHIBITOR RESPONSE1/ AUXIN SIGNALING F-BOX/AUXIN-RESISTANT/INDOLE-3-ACETIC ACID (TIR1/AFB-Aux/IAA) sensing module, downstream of which are SMALL AUXIN UP RNA (SAUR) proteins (Fendrych et al., 2016). Auxin induces the expression of SAUR. SAUR proteins are inhibitors of D clade type 2C protein phosphatases (PP2C.Ds), which dephosphorylate the penultimate Thr residue of PM H+-ATPases (Thr-947 in Arabidopsis AHA2) and inhibits PM H+-ATPase activity (Spartz et al., 2017; Ren et al., 2018). Auxin was also recently reported to rapidly induce transmembrane kinases (TMKs) interacting with PM H+-ATPases and activating PM H+-ATPases by direct phosphorylation at the penultimate Thr residue (Li et al., 2021; Lin et al., 2021). The TMK-dependent phosphorylation pathway and TIR1/AFB-SAUR-PP2C.D-mediated dephosphorylation pathway synergistically modulate auxin-induced PM H+-ATPase activation and cell expansion.

PM H+-ATPases contribute to nutrient uptake and transport. PM H+-ATPases generate proton gradients and electric potential differences across membranes, which energize secondary active transport to import nutrients into plant cells (Morales-cedillo et al., 2015). Phloem transport is an essential route of transport for most organic compounds. Immunohistochemical analysis revealed that PM H+-ATPases are highly expressed in the phloem (Villalba et al., 1991). In N. plumbaginifolia, the expression of a GUS reporter driven by the PMA4 promoter is detected in the phloem. Co-suppressed PMA4 transgenic N. plumbaginifolia plants display retarded growth and a male sterile phenotype, in addition to accumulating sucrose, fructose, and glucose in their leaves, indicating that this PM H+-ATPase affects the loading of organic compounds from leaves to the phloem (Zhao et al., 2000). Arabidopsis AHA3 localizes to phloem companion cells, which are rich in H+/sucrose cotransporters and mitochondria to energize local PM H+-ATPases with ATP (Ruth et al., 2014). Further work revealed that photosynthesis activates PM H+-ATPases by driving sugar accumulation (Okumura et al., 2016).

PM H+-ATPases also affect plant responses against pathogens. Plants detect pathogen-associated molecular patterns (PAMPs) through specialized pattern recognition receptors (PRRs), such as Flagellin Sensitive 2 (FLS2). The recognition of bacterial flagellin fragment flg22 by FLS2 activates intracellular responses to cope with the invading pathogen, such as a rise in cytosolic calcium (Ca2+) levels, a burst of reactive oxygen species (ROS), and apoplastic alkalization (Jeworutzki et al., 2010). At the same time, PM H+-ATPase activity is repressed (Keinath et al., 2010). Stomata, microscopic openings on the plant surface, are one of the possible entry points for bacteria and fungi (Melotto et al., 2006). One of the initial responses upon pathogen sensing is the closure of these stomatal pores, whereby flg22 causes changes in the phosphorylation status of AHA1 and AHA2. Phosphorylation of Thr-947 and Thr-881 in the C-terminal domain of AHA2 decreases, whereas phosphorylation of Ser-899 increases (Nühse et al., 2007). Phosphorylations at the Thr-947 and Thr-881 residues are required to activate PM H+-ATPases; by contrast, phosphorylation of Ser-899 inhibits proton transport (Fuglsang et al., 1999; Fuglsang et al., 2014; Haruta et al., 2014). Collectively, these changes in the PM H+-ATPases will repress its activity, ultimately leading to stomatal closure and preventing further pathogen invasion (Falhof et al., 2016).

Saline soil is a major threat to plant growth and crop yield (Zhou et al., 2013; Hu et al., 2017; van Zelm et al., 2020; Chen et al., 2022). More than 800 million hectares of the world’s land are affected by salinity (Munns and Tester, 2008). Soil alkalinity often co-occurs with salinity due to the abundance of sodium carbonate and sodium bicarbonate and aggravates the adverse effects caused by salinity on plant growth.

Plant tolerance against salt and alkaline stresses increases when improving PM H+-ATPase activity (Shi et al., 2000; Fuglsang et al., 2007; Yang and Guo, 2018a; Yang and Guo, 2018b). The Salt Overly Sensitive1 (SOS1) sodium transporter requires a proton gradient generated by PM H+-ATPases and is a key factor in response to salt stress (Qiu et al. 2002; Shen et al. 2011; Yang et al. 2019a). Overexpression of a constitutively activated form of PM H+-ATPase 4 (PMA4 lacking the autoinhibitory domain) in tobacco (Nicotiana tabacum) increases salt tolerance during germination and seedling growth, suggesting a correlation between PM H+-ATPase activity and salt tolerance (Gévaudant et al., 2007). The observed increased tolerance is mainly due to the posttranslational modification rather than a change in protein level (Morsomme and Boutry, 2000).

The regulatory role of PM H+-ATPases in response to saline-alkaline stress has been reported and summarized in Fig. 1. SOS2-like protein5 (PKS5), also named CBL-INTERACTING PROTEIN KINASE11 (CIPK11), is a negative regulator of PM H+-ATPase. Arabidopsis pks5 mutants exhibit high tolerance against alkaline stress and have high PM H+-ATPase activity. PKS5 negatively regulates the PM H+-ATPase AHA2 by phosphorylating the C terminus of the pump at Ser-931, thereby inhibiting the interaction between AHA2 and 14-3-3 (Fuglsang et al., 2007). In addition, inhibition of PM H+-ATPase activity in Saccharomyces cerevisiae is dependent on the presence of SOS3-like calcium-binding protein1 (SCaBP1, also named Calcineurin B-like protein2 [CBL2]). These results suggest that changes in Ca2+ signaling upon alkaline stress conditions regulate SCaBP1-PKS5-mediated PM H+-ATPase activity (Fuglsang et al., 2007). Another SOS2-like kinase, PKS24, interacts with SCaBP1 and negatively regulates PM H+-ATPase activity (Lin et al., 2014). In conclusion, SCaBP1-PKS5 and SCaBP1-PKS24 phosphorylate Ser-931 in AHA2 and blocking the interaction between AHA2 and 14-3-3. As a consequence, PM H+-ATPases are repressed under normal conditions (Fuglsang et al., 2007; Lin et al., 2014; Yang et al., 2019a). Another Ca2+ sensor, SCaBP3/CBL7, also interacts with AHA2 and prevents the interaction between AHA2 and 14-3-3, resulting in lower PM H+-ATPase activity under normal conditions. Furthermore, SCaBP3 enhances the interaction between PKS5 and AHA2, leading to increased PKS5-mediated suppression of PM H+-ATPase activity. Meanwhile, SOS2 kinase activity is inhibited by 14-3-3 (Zhou et al., 2014).

While under saline and alkaline stress, cellular Ca2+ level increases (Ma et al., 2019), resulting in SCaBP3 release from the AHA2 C terminus and alleviating the inhibitory interaction between the C terminus and the central loop. The activity of the SCaBP3-PKS5 complex and phosphorylation at Ser-931 decrease, allowing AHA2 to be activated by 14-3-3. Ca2+ also increases the interaction between 14-3-3 proteins and PKS5, which represses PKS5 activity, ultimately releasing the inhibition of SOS2 and AHA2 activity (Yang et al., 2019a; Yang et al., 2019b). DnaJ homolog 3 (J3) interacts with PKS5 to inhibit its kinase activity, thus alleviating the repression imposed on the PM H+-ATPases. j3 mutants are characterized by lower PM H+-ATPase activity and are hypersensitive to saline and alkaline stresses (Yang et al., 2010). Proton gradient generated by PM H+-ATPase AHA2 may enhance the activity of SOS1, an important Na+/H+ antiporter. Meanwhile, Ca2+ also promotes the interaction between SOS2, SOS3, and SCaBP8, then activates SOS1. Acetylation at Lys-56 of 14-3-3λ was recently shown to inhibit AHA2 activity and negatively regulate alkaline stress responses (Guo et al., 2022).

In addition, fatty acids and lipids have been reported to undergo dynamic changes under salt stress, such as the free unsaturated fatty acids oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), phosphatidycholine (PC), and lipid phosphatidic acid (PA) (Tasseva et al., 2004; Han et al., 2017; Li et al., 2019). Phosphatidylinositol (PI) was found to inhibit PM H+-ATPase activity under non-stress conditions in Arabidopsis. Under salt stress, PI is converted to phosphatidylinositol 4-phosphate (PI4P), which binds to and activates PM Na+/H+ antiporters to maintain ion homeostasis (Yang et al., 2021).

From the numerous examples above, it is clear that PM H+-ATPases are involved in salt and alkaline stress responses. However, the details of how plants sense the salt and alkaline stresses and lead to the regulation of PM H+-ATPases remains unclear.

Temperature is an important environmental factor affecting plant growth and development, and it dictates the geographical distribution of species. The plasma membrane is thought to be the primary site of cell response to low- and high-temperature stresses (Takahashi et al., 2013). Indeed, low temperatures cause changes in the lipid composition of the PM, leading to its rigidification. Conversely, higher temperatures are associated with the fluidization of the PM (Sadura et al., 2020). PM H+-ATPases are early injury sites during temperature stress and they are regulated in response to this abiotic stress (Arora and Palta, 1991). In sugar beet (Beta vulgaris) cell suspension cultures, cold stress (0–4°C) induces a large amount of cytoplasmic 14-3-3 proteins to bind to PM H+-ATPases to activate them (Chelysheva et al., 1999). A change in PM H+-ATPase activity in response to low temperature has also been reported in cucumber (Cucumis sativus) roots (Ahn et al., 2000; Janicka-Russak et al., 2012a). In Arabidopsis, PM H+-ATPase activity is affected differently depending on the duration of cold treatment. The proton pump activity is repressed upon cold (4°C) exposure for shorter periods, such as 6 h. However, during prolonged cold treatments (12–18 h), PM H+-ATPase activity recovers and dramatically increases. These changes in activity correlate with the extent of interaction between 14-3-3 and PM H+-ATPases. In addition, the transcription of the genes encoding PM H+-ATPases is also induced after long-term cold treatment, which also contributes to the increase in PM H+-ATPase activity (Muzi et al., 2016). A similar rise in PM H+-ATPase transcript levels and/or PM H+-ATPase protein abundance has been described under high temperature in pea (Pisum sativum), cucumber, and barley (Hordeum vulgare) (Liu et al., 2009; Sadura et al., 2020). Janicka-Russak et al. (2012b) reported that ABA and hydrogen peroxide stimulate the transcription of PM H+-ATPase genes and increase the phosphorylation of PM H+-ATPases to activate the enzyme.

Although many studies support the idea that PM H+-ATPases are key to temperature responses, how PM H+-ATPases are regulated under low and high temperatures is not fully understood. Seasonal changes in PM H+-ATPase activity and fatty acid (FA) composition were reported in red pine (Pinus resinosa) during cold acclimation and de-acclimation. This observation may support the hypothesis that FA-regulated PM H+-ATPase activity is involved in cellular responses to cold stress (Martz et al., 2006). Work by Sadura et al. (2020) also showed that growth temperature affects the accumulation of PM H+-ATPase transcripts and PM H+-ATPase proteins in barley leaves, possibly involving brassinosteroids in this regulation.

Agriculture has high water demands, such that drought stress and uneven distribution of freshwater resources will impose a major limitation on crop yield (Chen et al., 2021). Most of the water taken up by plants is lost through stomatal transpiration (Zhao et al., 2011; Qi et al., 2018; Zhao et al., 2018; Yang et al., 2022). The pairs of guard cells forming individual stomata swell or shrink to control the opening or closing of the pore, thereby regulating the amount of water loss from leaves (Zhao et al., 2016; Chen et al., 2020b). In Arabidopsis, all 11 AHA genes are expressed in guard cells, suggesting that PM H+-ATPases play an important role in stomatal function (Ueno et al., 2005). Blue light induces stomatal opening through the activating phosphorylation of PM H+-ATPases, while the phytohormone ABA is involved in stomatal closure under drought stress by regulating PM H+-ATPase activity, as detailed below (Shimazaki et al., 2007; Pei et al., 2022). ABA contributes heavily to plant responses to drought stress. Increased ABA contents under drought stress promote stomatal closure to reduce water loss (Cutler et al., 2010). ABA has been reported to negatively regulate PM H+-ATPases in Arabidopsis, which is required for ABA-induced stomatal closure under drought stress (Merlot et al., 2007). Two dominant mutants in AHA1 were identified based on their complete loss of ABA-mediated stomatal responses; these mutants, named open stomata2 (ost2), exhibit constitutively active PM H+-ATPase activity (Merlot et al., 2007). The mechanism by which ABA regulates PM H+-ATPase activity has been studied in some detail (Fig. 2). For example, ABA inhibits H+-ATPase activity by dephosphorylating the penultimate Thr (Thr-947 in AHA2) at the PM H+-ATPase C terminus in guard cells (Yin et al., 2013). The Snf1-related kinase 2.6 (SnRK2.6, also named OST1) is involved in this pathway (Merlot et al., 2007). Recently, Miao et al. (2021) reported that clade A protein phosphatases type 2C ABA-insensitive 1 (ABI1) interacts with the C terminus of Arabidopsis PM H+-ATPase AHA2 and dephosphorylates Thr-947 to inhibit the pump activity. In the Arabidopsis root system, ABA induces PM H+-ATPase phosphorylation by SnRK2.2 (and probably SnRK2.3), but not SnRK2.6, at an unknown inhibitory site (Planes et al., 2015). In addition to regulating PM H+-ATPase through phosphorylation and dephosphorylation, another mechanism to regulate PM H+-ATPase activity relies on Proton ATPase Translocation Control 1 (PATROL1) to recruit the H+-ATPases to the PM and affect stomatal movements (Hashimoto-sugimoto et al., 2013). VESICLE-ASSOCIATED MEMBRANE PROTEIN 711 (VAMP711) constitutes a third regulatory mechanism, whereby this N-ethylmaleimide-sensitive factor attachment protein receptor interacts with AHA1 and AHA2 at their C termini in response to ABA treatment and inhibits PM H+-ATPase activity. The vamp711 mutant displays higher PM H+-ATPase activity and slower stomatal closure under drought stress (Xue et al., 2018).

Potassium was also reported to play a critical role in plant responses to drought stress. Increased K+ uptake confers greater drought tolerance in Arabidopsis and in barley (Osakabe et al., 2013; Feng et al., 2016). Research by Cai et al. (2019) investigated K+ uptake in roots and the translocation from roots to shoots in barley under drought stress. The results suggested that PM H+-ATPase activity and/or expression of the encoding gene plays an important role in regulating the activity of K+ transporters and channels under drought stress.

Light is a vital environmental factor that greatly influences plant growth and reproduction. Light affects a series of physiological processes, including stomatal movement. Red light induces stomatal opening via photosynthesis, while blue light activates PM H+-ATPases to open stomata (Shimazaki et al., 2007; Kinoshita and Hayashi, 2011; Inoue et al., 2008) (Fig. 2).

Plant cells perceive blue light via the photoreceptor kinases, phototropin1 (phot1) and phototropin2 (phot2) (Kinoshita et al., 2001). Phototropins contain two domains: an N-terminal photosensory domain and a C-terminal serine-threonine kinase domain (Christie, 2007). The N-terminal domain contains two LOV (an abbreviation for light, oxygen, voltage) domains, LOV1 and LOV2. Upon excitation by blue light, the cofactor flavin mononucleotide (FMN) binds to a conserved Cys residue in the LOV domain, thereby alleviating inhibition of kinase activity by LOV2 (Crosson and Moffat, 2001). Blue light–activated phototropins then autophosphorylate and phosphorylate their substrate, Blue Light Signaling 1 (BLUS1), another protein kinase (Inoue et al., 2008; Takemiya et al., 2013a). The C terminus of BLUS1 acts as a regulatory domain for kinase activity, whereby phosphorylation at Ser-348 releases autoinhibition (Hosotani and Yamauchi, 2021). Activated BLUS1 subsequently activates an unknown kinase that phosphorylates the penultimate Thr in the PM H+-ATPases, creating a binding site for 14-3-3 to activate the pump (Kinoshita and Shimazaki, 2002; Kinoshita and Hayashi, 2011).

SAUR proteins have been reported to inhibit PP2C activity and activate PM H+-ATPases by promoting Thr-947 phosphorylation in the auxin-mediated cell expansion pathway (Spartz et al., 2017; Ren et al., 2018). The underlying model posited that the participation of BLUS1 activates PM H+-ATPases. Based on this model, BLUS1 may inactivate a protein phosphatase that then dephosphorylates the penultimate Thr in PM H+-ATPases, thus raising the phosphorylated state of the proton pump and enhancing its interaction with 14-3-3 to activate PM H+-ATPases under blue light (Falhof et al., 2016). SAUR and PP2C.D proteins were recently shown to act antagonistically to regulate stomatal opening via PM H+-ATPases and K+ transport (Wong et al., 2021).

Multiple other regulators function in this signaling pathway. For instance, the type 1 protein phosphatase PP1 and the Raf-like protein kinase BLUE LIGHT-DEPENDENT H+-ATPASE PHOSPHORYLATION (BHP) act downstream of BLUS1 and upstream of PM H+-ATPases (Takemiya et al., 2006; Hayashi et al., 2017). Similarly, PP1 REGULATORY SUBUNIT2-LIKE PROTEIN1 (PRSL1) functions as a regulatory subunit of PP1 (Takemiya et al., 2013b). The raf-like kinases CONVERGENCE OF BLUE LIGHT AND CO2 1 (CBC1) and CBC2 regulate the phosphorylation of guard cell PM H+-ATPases during blue light–mediated stomatal opening (Hiyama et al., 2017; Hayashi et al., 2020). Blue light–dependent PM H+-ATPase activation is inhibited by ABA through hydrogen peroxide–induced dephosphorylation of PM H+-ATPases (Zhang et al., 2004). However, it is still unclear how BLUS1 activates PM H+-ATPases under blue light, and the direct phosphorylation or dephosphorylation of PM H+-ATPases by key kinases or phosphatases needs to be determined. Besides blue light, red light–induced phosphorylation of PM H+-ATPases in leaves was also described and appeared to correlate with stomatal opening under red light (Ando and Kinoshita, 2018).

In an independent approach, library-based chemical screens identified new compounds that affect stomatal movement (Kinoshita et al., 2021). Nine chemical compounds, SCL1–SCL9 (for stomatal closing), were identified as suppressors of light-mediated stomatal opening in Benghal dayflower (Commelina benghalensis). SCL1 and SCL2 inhibit blue light–induced phosphorylation at the Thr-947 residue of PM H+-ATPases (Toh et al., 2018). Chemical screening of protease inhibitors (PIs) separately identified three inhibitors (PI1, PI2, and PI3, inhibitors of ubiquitin-specific protease 1, membrane type-1 matrix metalloproteinase, and matrix metalloproteinase-2, respectively) of blue light–induced phosphorylation of PM H+-ATPase (Wang et al., 2021a). Spraying SCL1 or PI1 onto leaves inhibits stomatal opening and decreases water loss in plants (Toh et al., 2018; Wang et al., 2021a). These chemicals may help reduce water needs in agriculture and prolong the shelf life of cut flowers (Kinoshita et al., 2021).

Aside from light quality, light intensity also affects PM H+-ATPases by changing its cellular accumulation pattern. 3D live-cell imaging revealed that light is required for the localization of Arabidopsis AHA2 to the plasma membrane. Under dim light, AHA2 localizes both to the intracellular compartments and in the plasma membrane at the transition zone between the meristem and the elongation zone in the root. Changes in localization are dependent on the receptor kinase FERONIA (FER) and cause apoplast alkalization, ultimately inhibiting root growth (Haruta et al., 2018).

Plants take up the nutrients they need to support growth from the soil. In fact, among the 17 essential elements required for plant growth and development, 14 mineral elements come from the soil. Nitrogen (N), phosphorus (P), and potassium (K) are macronutrients required in large quantities (Nath and Tuteja, 2015; Song et al., 2021). Nutrient deficiencies, especially those for N, P, and K, negatively affect agricultural production and yield (Guo et al., 2021; Wang et al., 2021b). PM H+-ATPases are involved in plant responses to nutrient deficiencies (Fig. 3). The proton gradient and electrical potential difference generated by PM H+-ATPases drive secondary active transport, which imports nutrients into plant cells and transports them in different cells or organelles (Morales-Cedillo et al., 2015).

Decades ago, a study revealed that PM H+-ATPases play a critical role in the generation of the electrical membrane potential driving P uptake (Ullrich-Eberius et al., 1981). PM H+-ATPase activity was later shown to be enhanced under low P stress (Shen et al., 2006; Chen et al., 2013). Work in tomato (Solanum lycopersicum) determined that 14-3-3 proteins in roots activate PM H+-ATPases experiencing low P conditions (Xu et al., 2012). AHA2 and AHA7 are highly expressed in Arabidopsis roots under P deficiency. Both encoded pumps mediating H+ efflux but in different parts of the root to achieve a distinct outcome: in the root elongation zone for AHA2, thus affecting primary root elongation, and in the root hair zone for AHA7 for the formation of root hairs (Yuan et al., 2017). In rice, the GTP-binding protein (G protein) γ-subunit qPE9-1 regulates root elongation to modulate P uptake via 14-3-3 proteins and PM H+-ATPases (Wang et al., 2021c).

Increased expression and/or activity of PM H+-ATPase is thought to positively correlate with activation of high-affinity phosphate transporters, such as the PHosphate Transporter 1 (PHT1) family, which takes up phosphate under low P stress (Siao et al., 2020), although direct evidence is still lacking to support this idea. In addition, organic acids (OAs) can be secreted into the rhizosphere and release the fixed phosphate from soil for plant uptake, and PM H+-ATPases contribute to this process (Crombez et al., 2019). P deficiency induces citrate exudation by increasing the activity of PM H+-ATPases in lupin (Lupinus pilosus) (Ligaba et al., 2004; Tomasi et al., 2009). Moreover, auxin levels and polar transport increase in roots under low P stress, thereby promoting root hair elongation (Giri et al., 2018). Transport of auxin via the AUXIN RESISTANT1 (AUX1) transporter is thought to require an H+ symporter. Auxin was reported to be involved in low P stress response by activating PM H+-ATPases (Shen et al., 2006), but more research is needed.

N constitutes 1.5%–8% of total dry weight in plants (Frink et al., 1999). Nitrate (NO3–) and ammonium (NH4+) are the two main N forms taken up by plants directly from the soil through root-specific transporters (Nath and Tuteja, 2015). PM H+-ATPase activity is induced under N deficiency and promotes NO3– and NH4+ uptake through nitrate transporters (NRTs) and ammonium transporters (AMTs) in maize (Zea mays) and rice (Santi et al., 2003; Zhang et al., 2021). Starvation and resupply of NO3– and NH4+ induce the expression of AHA2 in Arabidopsis and that of OSA2 and OSA7 in rice (Sperandio et al., 2020). Overexpression of OSA1 in rice leads to a 33% increase in grain yield and an overall 46% increase in N use efficiency (Zhang et al., 2021).

K itself amounts to 2%–10% of plant dry weight and plays an important role in various physiological processes for plant growth, development, and stress responses (Maathuis and Amtmann, 1999). Due to the high K+ concentration of the plant cytoplasm and the low K+ contents in the soil, the uptake of K+ by plants works against a steep concentration gradient, which requires the membrane potential generated by PM H+-ATPases as a driving force (Palmgren, 2001). K+ deficiency increases the activity of PM H+-ATPases, most likely AHA2 (Wang and Wu, 2013). One study showed that intracellular K+ can bind to the Asp-617 residue in the phosphorylation domain of PM H+-ATPases (Buch-Pedersen et al., 2006). K+ binding induces the dephosphorylation of E1P phosphorylated confirmation of the pump and negatively regulates PM H+-ATPases by uncoupling ATP hydrolysis and proton transport (Buch-Pedersen et al., 2006). K+ deficiency leads to a decrease in cytosolic K+, which may promote the activation of PM H+-ATPases (Wang and Wu, 2013). Activated PM H+-ATPases then cause hyperpolarization of the PM and extracellular acidification. PM hyperpolarization activates K+ channels, such as AKT1, and promotes K+ uptake under K+-deficient conditions (Fuchs et al., 2004). K+ deficiency–induced extracellular acidification can also energize K+ transporters, such as HIGH AFFINITY K+ TRANSPORTER5 (HAK5), to take up K+ (Philippar et al., 1999).

Acidic soils, defined as soils with a pH of 5.5 or lower, pose a threat to agricultural production worldwide. More than half of the world’s potential arable lands are acidic (von Uexküll and Mutert, 1995). Some ions accumulate in acidic soil and impact plant growth. Aluminum (Al) toxicity is a major limiting factor affecting plant growth in acidic soils (Zhang et al., 2017). Plants survive under Al stress by excluding Al from entry into the cytoplasm and/or detoxifying Al once inside cells (Ma, 2007). Organic acids play a critical role in Al exclusion. Al has been reported to induce the expression of the malate transporter gene Al-ACTIVATED MALATE TRANSPORTER (ALMT) and the citrate transporter gene MULTIDRUG AND TOXIC COMPOUND EXTRUSION (MATE) to facilitate the exudation of malate and citrate anions, which can chelate the Al cations present in the rhizosphere for detoxification (Hoekenga et al., 2006; Magalhaes et al., 2007). Notably, MATE-mediated citrate efflux is dependent on PM H+-ATPases, as they can provide protons and a charge gradient to drive MATE transporter activity (Zhang et al., 2017). Increased citrate exudation appears to be related to enhanced activity of PM H+-ATPase in lupin and carrot (Daucus carota) under P deficiency or Al stress (Ohno et al., 2003; Ligaba et al., 2004). Under Al stress in soybean (Glycine max) and fava bean (Vicia faba), Al-induced citrate efflux increases or decreases using the classic PM H-ATPase inducer (fucocomycin) or inhibitor (vanadate), respectively (Shen et al., 2005; Chen et al. 2013; Guo et al. 2013). In these studies, both the transcription of PM H+-ATPase and the protein abundance of PM H+-ATPase were enhanced in Al-resistant bean cultivars. Overexpression of PM H+-ATPase or chemical activation by known activators (such as magnesium or auxin) significantly increased plant tolerance against Al stress (Guo et al., 2013; Chen et al., 2015; Wang et al., 2016).

Further studies on the regulatory mechanism of PM H+-ATPases in response to Al stress revealed that phosphorylation of the penultimate Thr residue in PM H+-ATPases and the interaction between PM H+-ATPases and 14-3-3 increase in response to Al stress (Shen et al., 2005; Chen et al. 2013; Guo et al. 2013). Al treatment clearly raises PM H+-ATPase expression and increases the activity of the encoded proteins via phosphorylation to increase MATE-mediated citrate exudation, forming a nontoxic complex with Al. However, the key kinase that phosphorylates PM H+-ATPases upon exposure to Al stress remains unknown. Nitric oxide (NO) was recently shown to act upstream of hydrogen sulfide (H2S) to enhance the expression and activity of the PM H+-ATPase-coupled MATE transporter system, ultimately improving Al-induced citrate exudation (Wang et al., 2019) (Fig. 4). Notably, Al-induced NO generation depends on a nitrate reductase–mediated pathway in soybean roots (Wang et al., 2016), but the molecular identity of the various players in this pathway remains unknown.

Heavy metals in the soil can have a remarkable influence on plant growth and reproduction. Several heavy metals, such as iron, copper, and zinc, are essential trace elements at low concentrations but become toxic when present at high concentrations. Plants have no use for other heavy metals, such as cadmium, lead, or arsenium, which inhibit plant growth (Hall, 2002; Shi et al., 2015). The PM is thought to be the first target affected by heavy metal toxicity. The maintenance of ion homeostasis in plant cells under heavy metal stress requires a proton gradient generated by PM H+-ATPases.

Cadmium (Cd) disturbs many physiological processes, such as photosynthesis, respiration, and nutrient uptake (Jakubowska and Janicka, 2017). Cd first destroys PM structures and enhances membrane permeability. Short-term (2 h) exposure of cucumber seedling roots to Cd was reported to inhibit PM H+-ATPase activity (Janicka-Russak et al., 2008). Prolonged Cd treatment increases proton pump activity in rice and cucumber roots (Ros et al., 1992; Janicka-russak et al., 2012c). However, in oat (Avena sativa) and maize roots, long-term treatment with Cd leads to the inhibition of PM H+-ATPase activity (Astolfi et al., 2003; Astolfi et al., 2005). While these results appear to be contradictory, it is clear that PM H+-ATPase activity changes under Cd stress. The mechanisms by which PM H+-ATPases participate in Cd stress is not clear. In cucumber seedling roots, Cd increases both the expression of PM H+-ATPases and the activity of the encoded protein(s). Indeed, the transcription and phosphorylation levels of several PM H+-ATPases (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) are increased after six days of Cd treatment (Janicka-russak et al., 2012c). Jakubowska et al. (2015) reported that NADPH oxidases of the RBOH (Respiratory burst oxidase homolog) family are involved in the regulation of PM H+-ATPase activity under Cd stress. The model advanced by the authors posits that long-term Cd exposure increases the activity of NADPH-generating enzymes to produce more cytosolic NADPH, resulting in increased NADPH oxidase activity at the PM (Jakubowska et al., 2015). Superoxide radicals generated by NADPH oxidase can be dismutated into hydrogen peroxide, thus stimulating the expression of genes encoding PM H+-ATPases under Cd stress (Janicka-Russak et al., 2012c; Jakubowska et al., 2015). Later work revealed that brassinosteroids induce Cd stress tolerance through PM H+-ATPase and NADPH oxidase (Jakubowska and Janicka, 2017).

Copper (Cu) is another essential mineral element for plants but can be toxic at high concentrations (Hall, 2002). One of the most remarkable plant responses under Cu stress is rapid ion efflux (such as K+) caused by superfluous Cu-induced plasma membrane damage. The timely supplementation of these lost essential elements is important for survival during Cu toxicity. PM H+-ATPases energize secondary active transport and thus play a role in Cu tolerance. Studies have reported that the activity of PM H+-ATPase changes under Cu stress. As with Cd, PM H+-ATPase activity is suppressed by Cu2+ in vitro and in cucumber roots following a 2-h treatment (Janicka-Russak et al., 2008), but increased with a longer exposure to Cu (Janicka-russak et al., 2012c). Increased PM H+-ATPase activity in plants under Cu stress is associated with higher phosphorylation levels of the enzyme, which increases its interaction with 14-3-3 proteins to activate PM H+-ATPases (Janicka-russak et al., 2012c). The exact mechanism by which plants respond to heavy metal toxicity by regulating PM H+-ATPase is not fully understood. More studies are needed to elucidate this issue and improve plant tolerance to heavy metal toxicity in the future.