Membrane transporters, including ion channels and carriers, maintain ionic and nutrient homeostasis vital for growth and stress resilience in plants. Their activity is finely regulated, enabling dynamic responses to internal and external signals.

Transporters often exist in an 'autoinhibited' resting state that can be switched on by post-translational modifications in response to environmental cues. Specifically, protein phosphorylation functions as a molecular switch that relieves autoinhibition of channels and carriers to modulate their activities. We examine the phospho-regulation of exemplary transporters involved in nutrient allocation, stress resistance, and signal transduction, providing a perspective on how plants orchestrate membrane transport processes to sustain ion and nutrient balance and adapt to environmental challenges.

Cell membrane represents a critical barrier with limited permeability that defines the boundary between the intracellular and extracellular environments. In a primitive cell, the primary function of a cell membrane was to protect the internal environment from the outside, maintaining a clear separation with minimal exchange of substances. As life became more complex, cellular demands drove the evolution of more sophisticated systems for selective and dynamic exchange of molecules. To meet these demands, a diverse array of membrane transporters (see Glossary) emerged, each specialized to facilitate the movement of certain ions, nutrients, and other molecules across the membrane [1,2]. These transport proteins operate under tight regulation, ensuring that the flux of substances into and out of the cell is precisely controlled to meet the metabolic needs and respond to environmental conditions [2,3]. Among these transporters, ion carriers and channels play pivotal roles in maintaining cellular ionic homeostasis. Ion carriers, including pumps and exchangers, often rely on energy sources such as adenosine triphosphate (ATP) and membrane potential to actively transport ions against their electrochemical gradients. These carriers are essential for generating and maintaining the ionic gradients that are critical for various cellular processes. By contrast, ion channels typically facilitate the passive movement of ions down the electrochemical gradients, contributing to rapid changes in membrane potential and intracellular ion concentrations. At the single-cell level in eukaryotes, these transport proteins not only mediate the movement of ions across the plasma membrane but also distribute them among distinct organelles within the cell. This intracellular ion distribution is crucial for various cellular functions, including metabolism, signaling, and maintaining the structural integrity of organelles. The activities of ion transporters are tightly regulated to optimize ionic concentrations for cellular needs. This regulation is crucial for the transport of nutrient ions that must be adjusted according to metabolic demand and external supply, and for toxic ions that are carefully partitioned to prevent cellular damage [4].

Membrane transport proteins in plants are regulated by autoinhibition and phosphorylation, ensuring precise ionic homeostasis and nutrient distribution for growth and stress adaptation.

Phosphorylation modulates plasma membrane proton-ATPases (AHAs), potassium transporters (AKT1, HAK5), and nitrate and ammonium transporters (NRTs, AMTs), optimizing uptake, distribution, and ionic balance under fluctuating supply.

Phospho-regulation of the sodium transporter SOS1 enhances salt tolerance, while the anion channels SLAC1 and ALMT4 and the vacuolar transporter CLCa regulate stomatal closure to strengthen drought tolerance.

Calcium signaling is orchestrated through phosphorylation of influx channels [Cyclic Nucleotide-Gated Channels (CNGCs), OSCAs] and efflux pumps [autoinhibited Ca 2+ -ATPases (ACAs), Ca 2+ /H + exchangers (CAXs)], finetuning calcium dynamics critical for adaptation and development.

The regulation of ion transporters involves a complex integration of internal and external signals. Internal signals, such as those generated by metabolic activity, and external signals, such as the availability of ions in the extracellular environment, are processed by sophisticated signaling networks. These networks connect cellular signals to the control of transporter activity at multiple levels, including transcriptional, post-transcriptional, and post-translational modifications [5][6][7][8]. While transcriptional regulation can adjust the protein level of ion transporters, post-translational modifications can offer a mechanism that rapidly alters transporter activity in response to changing conditions. Among post-translational modifications, phosphorylation plays a central role in modulating transporter activity. To elucidate these phosphorylation-dependent regulatory mechanisms, researchers use multiple complementary approaches. Phospho-proteomics using high-resolution mass spectrometry enables the global identification of phosphorylation sites on numerous proteins extracted from specific tissues of plants under various growth conditions or in response to a specific treatment. Biochemical approaches, such as in vitro kinase assays, protein-protein interaction analyses, and site-directed mutagenesis, can connect kinases with their substrates and specific phosphorylation sites. Genetic approaches can explore the functional relevance of specific sites on protein substrates and their kinases in a physiological context. These approaches provide a general pipeline for dissecting the molecular basis of phosphorylation-dependent regulation of plant proteins, including ion transporters.

In this review, we focus on the dynamic regulation of ion transporters in plant cells, emphasizing mechanisms such as autoinhibition and phospho-regulation. While autoinhibition serves as an intrinsic regulatory mechanism to maintain transporters in an inactive state, phosphorylation often functions to relieve this autoinhibition and enhance transporter activity. Such autoinhibitory control is not unique to transporters; it reflects a general regulatory strategy observed across diverse proteins in all domains of life, where activation commonly results from the removal of inhibitory mechanism [9]. Together, these mechanisms provide rapid and reversible control, enabling plant cells to fine-tune transport functions in response to both internal metabolic cues and external environmental changes. We highlight how these mechanisms operate as dynamic gating systems, orchestrating ion homeostasis to support cellular functions.

Efficient nutrient acquisition and utilization are fundamental processes for plant growth and productivity. Ion channels and carriers play pivotal roles in mediating the uptake and distribution of essential mineral nutrients across cellular membranes. Phospho-regulation is a universal mechanism in modulating the activity of these proteins, ensuring precise control of nutrient homeostasis in response to developmental cues and environmental fluctuations.

The plasma membrane proton (H + ) pumps (H + -ATPases; referred to as AHAs for autoinhibited H + -ATPases) are central to plant nutrient uptake by establishing the H + motive force and a negative membrane potential, which drive secondary transport via proton-coupled symporters, antiporters, and channels [10,11]. Autoinhibition of plant H + -ATPases was first revealed by proteolytic removal of a small C-terminal segment leading to a strongly activated enzyme. Such a finding identified the C-terminal region as an autoinhibitory domain, and later studies suggested that phosphorylation may relieve the autoinhibition under physiological conditions [12,13]. More recent work in arabidopsis (Arabidopsis thaliana) elucidated the detailed phospho-regulation mechanism of the plasma membrane H + -ATPase, with AHA2 serving as the best-studied isoform [14]. Phosphorylation at the penultimate threonine residue (Thr-947) of AHA2 acts as a critical regulatory switch, mediating its interaction with 14-3-3 proteins. This interaction stabilizes the enzyme in its active conformation, significantly enhancing H + -pumping activity [15]. Further analyses indicate that phosphorylation of multiple sites can occur in the autoinhibitory domain and may

Autoinhibition: an intrinsic regulatory mechanism where a transport protein remains in an inactive state due to specific structural constraints or interactions. Ca 2+ -ATPases: ATP-driven calcium pumps that actively remove Ca 2+ from the cytosol into intracellular compartments or the extracellular space. Ca 2+ -dependent protein kinase: a family of protein kinases that integrate Ca 2+ signaling with protein phosphorylation, playing crucial roles in development, stress responses, and membrane transport regulation. Ca 2+ /H + exchangers: secondary active transporters that remove Ca 2+ from the cytosol by exchanging it for H + across membranes. Calmodulin (CaM): a highly conserved Ca 2+ -binding protein that acts as a sensor and mediator of Ca 2+ signaling in eukaryotic cells. Upon Ca 2+ binding, calmodulin undergoes conformational changes to regulate various target proteins, including kinases, ion channels, and transcription factors. CBL-CIPK network: a calcium signaling pathway that involves calcineurin B-like (CBL) proteins and their interacting kinases (CIPKs), which regulates various ion transporters and stress responses. Cyclic nucleotide-gated channel (CNGC): ion channels that mediate fluxes of Ca 2+ and other cations, with activation by cyclic nucleotides like cAMP and cGMP well-established in animal channels but not in plant channels. Ion homeostasis: the balance of intracellular and extracellular ion concentrations maintained by transport proteins to support cellular functions and adaptation to environmental changes. Membrane transporters: proteins, including channels and carriers, that facilitate the movement of ions and molecules across cellular membranes. Mildew resistance locus O: a plant gene family encoding transmembrane proteins that negatively regulate immunity against powdery mildew fungi. Emerging evidence suggests that MLOs may function as Ca 2+ -permeable channels, linking calcium signaling to broader physiological processes such as pollen tube growth, pollen reception, and stress adaptation.

either enhance or suppress the enzyme activity, depending on the specific residues phosphorylated [16][17][18][19]. In addition to driving secondary transport, phospho-regulation of AHAs allows plants to dynamically modulate rhizosphere acidification vital for the solubilization and uptake of poorly mobile nutrients such as phosphate [20] and iron [21].

OSCA/CSC: OSCA (Reduced Hyperosmolality-Induced Ca 2+ Channel) and CSC (Calcium-Permeable Stress-Gated Cation Channel) are mechanosensitive ion channels that mediate Ca 2+ influx in response to osmotic and mechanical stress. They can be activated by membrane tension or osmotic changes, playing key roles in early stress signaling. Phospho-regulation: control of protein activity through phosphorylation, a reversible post-translational modification of proteins by the addition and removal of phosphate groups by protein kinases and phosphatases. Proton (H + ) pumps: membrane transport proteins with ATPase activity that actively move protons (H + ) across cellular membranes using ATP hydrolysis to generate electrochemical gradients. Rapid alkalinization factor: a family of small peptides that regulate cell growth and signaling by modulating ion transport and extracellular pH. Receptor-like cytoplasmic kinase (RLCK): a large group of kinases that often function downstream of receptorlike kinases to mediate intracellular signaling in response to extracellular stimuli. Receptor-like kinase (RLK): a large family of membrane-bound kinases that perceive extracellular signals and activate intracellular signaling cascades, regulating diverse processes including development, immunity, and environmental adaptation. Secondary messenger: a signaling molecule, such as calcium Ca 2+ , that relays cellular signals from upstream receptors or sensors to target proteins, amplifying cellular responses. Slow anion channel: a family of anionselective channels that mediate fluxes of anions such as chloride and nitrate across membranes. SNF1-related protein kinase 2: a family of kinases that play a central role in abscisic acid signaling and stress responses in plants.

Studies on potassium (K + ) uptake further highlight a critical role of phospho-regulation in nutrient acquisition. K + is an essential macronutrient involved in osmoregulation, enzyme activation, and membrane potential maintenance [22]. Under K + -deficient conditions, plants mobilize multiple transport systems to optimize K + uptake, primarily through phospho-regulation of AKT1 and HAK5 [23,24]. AKT1, an inward-rectifying K + channel in the plasma membrane, is vital for K + uptake, especially when plants face low K + availability [25]. Its activity is strongly enhanced by phosphorylation through the signaling complex consisting of calcineurin B-like proteins 1 and 9 (CBL1 and 9) and CBL-interacting protein kinase 23 (CIPK23). In this pathway, calcium (Ca 2+ ) binds to CBL1/9 proteins which activate the kinase CIPK23 and recruit it to the plasma membrane, leading to AKT1 phosphorylation and increased K + transport [26][27][28]. Interestingly, under severe K + deficiency, the high-affinity transporter HAK5 is also swiftly phosphorylated and activated by the CBL1/9-CIPK23 complex, further enhancing K + uptake [29]. Using structural modeling based on other K + transporters and functional assays in yeast, HAK5 was proposed to be activated through N-terminal phosphorylation while its C-terminal tail serves as an autoinhibitory domain, providing a mechanistic framework for its conditional activation under low-K + conditions [30]. In addition to plasma membrane transporters, vacuolar tandem-pore K + (TPK) channels maintain cytosolic K + balance via K + release from vacuolar stores [31]. Recent studies revealed that under low-K + conditions, Ca 2+ signals activate the vacuolar CBL2/3-CIPK3/9/26 signaling pathway, leading to the phosphorylation and activation of tonoplast-localized TPK1/3/5 channels, which facilitate vacuolar K + remobilization to enable plant adaptation to K + scarcity [32]. Collectively, the concerted phospho-regulation of plasma membrane AKT1, HAK5, and tonoplast TPKs constitutes a versatile and efficient response to fluctuating K + levels, supporting K + homeostasis and plant growth [33] (Figure 1A).

Extending the theme of phospho-regulation in nutrient transport, nitrogen (N) uptake through nitrate (NO 3 -) and ammonium (NH 4 + ) transporters is also fine-tuned by phosphorylation to adapt to changing N availability. This is exemplified by the dual-affinity NO 3 -transporter NRT1.1/CHL1 that mediates NO 3 -uptake and acts as a NO 3 -sensor in plants [34]. Phosphorylation at Thr-101 switches NRT1.1 between low-and high-affinity states, allowing dynamic adaptation to fluctuating NO 3 -availability [35]. When external NO 3 -levels are low, phosphorylation at Thr-101 by the CBL9-CIPK23 kinase complex shifts NRT1.1-mediated transport to a high-affinity mode, enhancing NO 3 -uptake. Under high NO 3 -conditions, the unphosphorylated form favors low-affinity transport. Plants utilize NRT1.1 as both a transporter and a sensor to modulate NO 3 -uptake and coordinate signaling pathways in response to soil NO 3 -fluctuations [34]. In contrast to NO 3 transporters, NH 4 + transporters (AMTs) are primarily regulated through inhibitory phosphorylation to prevent excessive NH 4 + uptake and toxicity [36]. Notably, the same CBL1/9-CIPK23 signaling complex that activates NRT1.1 (CHL1) under low NO 3 -conditions exerts an opposite effect on AMT1 transporters [37]. Under high external NH 4 + levels, CIPK23 phosphorylates AMT1;1 and AMT1;2 at conserved threonine residues in their C-terminal regions, leading to their inactivation [38]. The positive and negative regulation of NO 3 -and NH4 + uptake by the same kinase enable plants to prioritize accumulation of NO 3 -over NH4 + to avoid NH4 + cytotoxicity [39,40]. This example also highlights that, while many phosphorylation events enhance transporter activity, some can suppress transport as part of a negative regulation. The dual roles of phosphorylation, both activating and inhibitory, allow transporters to operate as finely tunable molecular machines in maintaining cellular homeostasis.

Figure 1. Phospho-regulation of ion transporters in plant nutrient uptake and abiotic stress responses.

(A) Phospho-regulation of potassium (K + ) channels and transporters under K + scarcity. Low K + availability may activate Ca 2+ influx through an unidentified Ca 2+ channel, elevating cytosolic Ca 2+ levels. This Ca 2+ signal is perceived by CBL proteins, which recruit and activate CIPKs to phosphorylate their target transporters. At the plasma membrane, the CBL1/ 9-CIPK23 complex phosphorylates and activates AKT1 and HAK5, enhancing K + uptake into the cytosol. In the vacuolar membrane, the tonoplast-localized CBL2/3-CIPK9 complexes phosphorylate and activate tandem-pore K + (TPK) channels, promoting K + release from the vacuole to support cytosolic K + homeostasis. (B) Phospho-regulation of NRAMP1 and IRT1 in manganese (Mn 2+ ) and iron (Fe 2+ ) uptake. Under Mn 2+ or Fe 2+ deficiency, Ca 2+ influx activates CPK21 and CPK23, which phosphorylate NRAMP1 in the plasma membrane to facilitate Mn 2+ uptake and IRT1, a highaffinity Fe 2+ transporter, to enhance Fe 2+ absorption. This phosphorylation-mediated regulation optimizes metal ion acquisition under nutrient-limiting conditions. (C) Phospho-regulation of BOR1 under boron deficiency. Boron scarcity induces Ca 2+ signaling and activation of CPK10 that phosphorylates BOR1, a borate exporter in the plasma membrane. This modification enhances BOR1-mediated boron loading into the xylem for root-to-shoot translocation, elevating boron supply under deficiency conditions. (D) Phospho-regulation of Na + transporters during salt stress. High salinity triggers Ca 2+ signaling, leading to elevated cytosolic Ca 2+ levels and activation of CBL-CIPK pathways that regulate Na + homeostasis. In the plasma membrane, the CBL4/SOS3-CIPK24/SOS2 complex phosphorylates SOS1, a Na + /H + antiporter, enhancing Na + extrusion to prevent toxic Na + accumulation in the cytosol. At the vacuolar membrane, the CBL10-CIPK24 complex phosphorylates an unidentified Na + transporter, promoting Na + sequestration into the vacuole to mitigate cytosolic Na + toxicity. (E) Phospho-regulation of SLAC1, CLCa, and ALMT4 in stomatal closure during drought stress. Under drought conditions, abscisic acid (ABA) accumulates and inhibits ABI1, a negative regulator of kinase activity. This de-repression allows OST1, CPK21, and CPK23 to phosphorylate and activate SLAC1, a slow anion channel in the guard cell plasma membrane. Activated SLAC1 facilitates Cl -efflux, driving membrane depolarization and promoting stomatal closure to reduce water loss. In addition, the tonoplast-localized CLCa is phosphorylated, which modulates the directionality of NO 3 -/H + exchange across the vacuolar membrane, thereby fine-tuning vacuolar anion storage and release. ALMT4, another vacuolar anion transporter, is phosphorylated to mediate malate release, coordinating cytosolic and vacuolar anion balance during stress adaptation. In all panels, phosphorylation events are indicated by a capital letter 'P' inside a pink circle and black arrows denote the direction of ion movement.

Phospho-regulation of plant transporters also plays a critical role in managing uptake of micronutrients, such as manganese (Mn), iron (Fe), and boron (B). Studies have identified CPK21 and CPK23, two Ca 2+ -dependent protein kinases (CDPKs; CPKs), as key regulators of Mn and Fe homeostasis. In the case of Mn, CPK21 and CPK23 phosphorylate the Mn 2+ transporter NRAMP1, enhancing its activity to support Mn 2+ uptake and distribution under deficiency [41]. Likewise, under Fe-limited conditions, these two kinases phosphorylate and activate the Fe 2+ transporter IRT1, boosting Fe 2+ acquisition to meet cellular demands [42] (Figure 1B). In addition, BOR1 serves as the key transporter for boron, mediating its xylem loading and long-distance translocation essential for fertility and shoot development [43]. Recent work showed that CPK10 phosphorylates and activates BOR1, thereby enhancing tolerance to boron deficiency [44]. Collectively, the phosphorylation of NRAMP1, IRT1, and BOR1 exemplifies a unifying post-translational mechanism that harnesses Ca 2+ signaling to control micronutrient transporters, enabling plants to fine-tune nutrient uptake and adapt to changing nutrient conditions (Figure 1C).

Plants constantly encounter environmental stresses such as high salinity and drought, which threaten their survival and productivity. To cope with these challenges, plants employ regulatory networks to modulate the activity of ion transporters, enabling rapid adaptation. In these regulatory networks, phosphorylation, again, serves as a dynamic and reversible switch, fine-tuning transport activities in response to environmental cues.

High salinity induces ionic stress in plants by causing toxic accumulation of sodium ions (Na + ). To counter this challenge, plants initiate the salt overly sensitive (SOS) pathway in which the Ca 2+dependent CBL4/SOS3-CIPK24/SOS2 kinase complex phosphorylates and activates SOS1, driving Na + efflux [45,46]. The Na + /H + exchanger SOS1 is in an autoinhibited state without salinity stress and salt-induced phosphorylation of SOS1 switches on its transport activity to expel Na + out of the cell, enhancing salt tolerance. In parallel, the CBL10-CIPK24/SOS2 signaling pathway also responds to salt stress by targeting an unidentified transporter localized in the tonoplast [47]. Phosphorylation of this vacuolar transporter by CIPK24/SOS2 is thought to facilitate the sequestration of excess Na + into vacuoles, which further protects the cell from Na + toxicity [48]. Interestingly, a recent study suggests that SOS1 is also localized to the tonoplast and contributes directly to vacuolar Na + sequestration, indicating a dual role for SOS1 in both Na + extrusion and intracellular compartmentalization [49]. It will be interesting to examine whether SOS1 is a target for the vacuolar CBL10-CIPK24 signaling pathway (Figure 1D).

During drought stress, plants efficiently conserve water by closing their stomata, pores in the leaf surface responsible for gas exchange [50]. Stomatal movement is associated with activities of multiple ion channels in the two guard cells that form stomata. Many of these ion channels are modulated by phosphorylation to control ion fluxes and change turgor pressure essential for stomatal opening and closure [51]. Central to this process is the slow anion channel 1 (slow SLAC1), which mediates the efflux of anions such as chloride (Cl -) from guard cells, leading to stomatal closure [52]. This process is controlled by reversible phosphorylation, integrating action of several kinases in response to the stress hormone abscisic acid (ABA). During drought stress, elevated ABA levels activate the SNF1-related protein kinase 2.6 (SnRK2.6), also known as open stomata 1 (OST1), which directly phosphorylates and activates SLAC1, initiating anion efflux and stomatal closure, whereas type 2C protein phosphatases (PP2Cs) dephosphorylate SLAC1 to provide reversible regulation once stress conditions subside [53][54][55]. Concurrently, OST1 also targets several cyclic nucleotide-gated channel (CNGC)-type Ca 2+ channels to elicit Ca 2+ elevation in guard cells to activate CDPKs, such as CPK3 and CPK21, which phosphorylate distinct sites on SLAC1 and lead to activation of the channel, linking Ca 2+ signaling to stomatal control [55][56][57]. Additionally, CBL1/9-CIPK23, part of the CBL-CIPK network, also phosphorylates SLAC1, contributing to guard cell regulation [58,59]. This multi-kinase phospho-regulation ensures precise and dynamic control of SLAC1, allowing plants to rapidly adjust stomatal aperture for optimal water conservation during drought stress, while enabling timely stomatal reopening for gas exchange and photosynthesis under favorable conditions, thereby balancing plant adaptation to stress and rapid growth in optimal environments (Figure 1E).

Beyond SLAC1, phosphorylation also modulates vacuolar anion transporters that contribute to guard-cell turgor regulation during stomatal closure. The vacuolar NO 3 -/H + exchanger CLCa exhibits phosphorylation-dependent changes of its transport properties, shifting between inwardand outward-rectifying behaviors and thereby altering vacuolar anion fluxes for turgor adjustment [60]. Similarly, ALMT4, a vacuolar malate channel, is activated in a phosphorylation-dependent manner to promote vacuolar anion efflux during ABA-induced stomatal closure [61]. Together, these vacuolar transporters coordinate with SLAC1 at the plasma membrane, forming a multilayered phosphorylation network that integrates hormonal and stress signals to drive efficient stomatal closure during drought stress (Figure 1E).

In addition to ion transport, regulated water movement across membranes is vital for stomatal closure during drought stress [62]. Plasma membrane intrinsic proteins (PIPs), particularly PIP2;1, facilitate water flux in guard cells, controlling turgor pressure and stomatal dynamics. Similar to the regulation of SLAC1, PIP2;1 is phosphorylated by OST1 at a specific serine residue (Ser-121) under drought conditions, enhancing its water transport activity [63]. This phosphorylation-driven activation of PIP2;1 works in concert with SLAC1-mediated anion efflux to rapidly adjust guard cell turgor, enabling efficient stomatal closure for water conservation during drought stress.

Beyond these well-established examples, phospho-proteomic studies have identified numerous phosphorylation sites in ion transporters during plant stress responses [64][65][66][67][68][69]. These largescale datasets offer a valuable resource for uncovering novel regulatory mechanisms underlying ion homeostasis and stress adaptation.

Ca 2+ is not only an essential plant nutrient but also a pivotal signaling molecule in plant responses to a wide array of developmental and environmental cues. Acting as a dynamic secondary messenger, transient fluctuations in cytosolic Ca 2+ concentrations encode specific signals, known as Ca 2+ signatures, which are decoded to trigger targeted physiological responses [70][71][72][73][74][75]. The generation of these Ca 2+ signatures relies on the finely tuned activities of Ca 2+ channels and carriers, which orchestrate Ca 2+ influx and efflux across cellular membranes [76,77]. This stringent control is essential because sustained high levels of cytosolic Ca 2+ are toxic to plant cells. To mitigate this risk, Ca 2+ influx channels must remain inactive under resting states, thereby limiting Ca 2+ entry to minimal levels. In response to a stimulus, a cell must initiate a rapid activation of Ca 2+ entry pathways to generate a cytosolic Ca 2+ elevation beyond a threshold level [70,76]. Equally important is the timely deactivation of these Ca 2+ channels to terminate the signal, assisted by prompt activation of Ca 2+ -scavenging systems to restore basal Ca 2+ levels [78,79]. During Ca 2+ signaling processes, autoinhibition and phospho-regulation are among the most common and effective mechanisms controlling Ca 2+ channels and carriers. These regulatory strategies provide rapid, reversible, and energy-efficient control, ensuring Ca 2+ signaling as both highly responsive and meticulously maintained to prevent prolonged Ca 2+ elevation that could compromise cell viability (Figure 2). (A) Phospho-regulation maintains cytosolic Ca 2+ homeostasis through vacuolar sequestration. Environmental Ca 2+ fluctuations require a housekeeping mechanism to prevent excessive cytosolic Ca 2+ accumulation. CNGC2/4 in the plasma membrane mediate Ca 2+ influx from the environment, increasing cytosolic Ca 2+ levels. Elevated cytosolic Ca 2+ binds to CBL2/3, which activates CIPK3/9/26 in the tonoplast. These kinases phosphorylate and activate the Ca 2+ /H + exchangers CAX1/3, which are otherwise autoinhibited, promoting Ca 2+ sequestration into the vacuole. This phosphorylation-dependent regulation provides a rapid response to fluctuations in external Ca 2+ , ensuring cytosolic Ca 2+ homeostasis. (B) Phospho-regulation of Ca 2+ transporters orchestrates Ca 2+ during stress adaptation. For instance, upon immune activation, multiple Ca 2+ transport pathways are phosphorylated to modulate Ca 2+ dynamics. Pattern recognition receptors (PRRs) interact with co-receptors BAK1/SERK4 and inhibit BTL2, while BTL2 positively regulates CNGC19/20, facilitating Ca 2+ influx into the cytosol during autoimmune responses. The PRR-BAK1 complex also phosphorylates BIK1/ PBL1, which in turn phosphorylates and activates multiple Ca 2+ channels, including CNGC2/4 and OSCA1.3/1.7 in the plasma membrane, to initiate Ca 2+ entry, triggering Ca 2+ signaling in different cell types. In parallel, BIK1/PBL1 phosphorylates CAX1/3 in the vacuolar membrane to promote Ca 2+ sequestration, shaping precise Ca 2+ transients and ensuring timely termination of the signal. Additionally, ACA8, a Ca 2+ -ATPase, mediates Ca 2+ efflux in the plasma membrane, with CIPK9 and CPKs potentially modulating its activity via phosphorylation. In all panels, phosphorylation events are indicated by 'P' inside a pink circle and black arrows denote the direction of Ca 2+ flow. Dashed line arrows indicate putative regulation.

Phospho-activation of Ca 2+ influx channels to spark cell signaling The initiation of Ca 2+ signaling hinges on the precise activation of Ca 2+ influx channels that mediate rapid Ca 2+ entry into the cytosol. These channels must remain tightly controlled, opening only in response to specific signals to generate distinct Ca 2+ signatures. This transient activation is crucial to prevent cytotoxic Ca 2+ overload and to maintain high sensitivity for future signal stimulations. Multiple Ca 2+ influx channels in plant cells are activated by phosphorylation to initiate Ca 2+ signal transduction. Different stimuli may integrate distinct channels to generate diverse Ca 2+ signatures decoded by various signaling pathways, leading to specific physiological responses.

Plant CNGCs are essential for mediating Ca 2+ influx in responses to various stimuli, including plant pathogens [80]. In arabidopsis, the CNGC2-CNGC4 heterotetramer forms a functional Ca 2+ channel that remains inactive in the resting state due to calmodulin (CaM) binding.

Upon detection of pathogen-associated molecular patterns (PAMPs) such as flagellin, the receptor-like cytoplasmic kinase (RLCK) BIK1, a core component of the pattern-triggered immunity (PTI) pathway, phosphorylates CNGC4. This phosphorylation likely induces a conformational change that disrupts CaM binding, thereby activating the CNGC2-CNGC4 complex and leading to Ca 2+ -dependent immune signaling [81]. A similar phosphorylation-dependent mechanism was observed in rice, where a PTI-related RLCK phosphorylates OsCNGC9, enhancing its activity to trigger Ca 2+ influx and downstream defense responses [82]. Besides plant immunity, a recent study revealed that CPK1 directly phosphorylates CNGC5/6/9, activating these channels to promote Ca 2+ influx required for root hair growth in arabidopsis [83]. This highlights a broader role of kinase-mediated phosphorylation in regulating CNGCs during different physiological processes. In contrast to phospho-activation, some CNGCs, such as CNGC19 and CNGC20, can be degraded after phosphorylation by the receptor-like kinase (RLK) BAK1 and SERK4 without directly affecting channel activity. Rapid turnover of these CNGC channels prevents prolonged Ca 2+ influx and subsequent cell death [84]. However, during enhanced immunity response (hypersensitive response), cell death is triggered by activation of CNGC20 through phosphorylation by another receptor kinase BTL2 [85]. Collectively, these findings highlight phosphorylation as a conserved yet versatile mechanism for modulating CNGC function, enabling plants to delicately regulate Ca 2+ influx for effective responses that either maintain cellular homeostasis or induce cell death, depending on the specific signals they receive.

In addition to CNGCs, the OSCA/CSC family of Ca 2+ -permeable channels also plays a critical role in plant immunity through phosphorylation-mediated regulation. Specifically, OSCA1.3 has been identified as a component in stomatal defense responses. Upon pathogen detection, BIK1 directly interacts with and phosphorylates OSCA1.3 in the N terminus, which activates the channel and facilitates Ca 2+ influx [86]. The resulting Ca 2+ signal triggers stomatal closure to limit pathogen entry. Notably, BIK1 functions as a central kinase that can simultaneously phosphorylate both CNGCs and OSCAs, providing temporal and spatial control of Ca 2+ signaling necessary for precise immune responses [87].

Certain plant Ca 2+ channels may depend on phosphorylation for proper function, though the underlying mechanisms remain unclear. For example, arabidopsis glutamate receptor-like (GLR) channels, such as GLR3.6 and GLR3.7, are phosphorylated by CDPKs, facilitating their interaction with 14-3-3 proteins and regulating root growth under salt stress [88,89]. While these studies implicate phosphorylation as a regulatory mechanism, the specific impact on ion transport and its broader physiological relevance remain to be fully understood. Mildew resistance locus O (MLO) proteins have recently been identified as a new class of plant Ca 2+ channels, one of which forms a receptor-channel complex that mediates Ca 2+ signaling during pollen tube reception [90]. Further studies reveal that the rapid alkalinization factor (RALF) signaling pathway activates several MLO channels to maintain pollen tube integrity. In this pathway, MLO1/5/9/15 channels are gated by the RLKs BUPS1/2 in complex with the RLCK MARIS, enabling the Ca 2+ influx to control pollen tube growth [91]. Although direct phosphorylation of MLO channels by the BUPS1/2-MARIS kinase complex has yet to be demonstrated, the activity of MLO Ca 2+ channels in this context clearly depends on the kinase complex, highlighting phosphorylation as a likely regulatory mechanism.

Phospho-regulation of calcium efflux transporters for signal termination and homeostasis While activation of Ca 2+ influx channels initiates signaling cascades, timely removal of cytosolic Ca 2+ is equally important to reset the resting state to maintain homeostasis and avoid cytotoxicity. This essential step is predominantly facilitated by two major classes of Ca 2+ efflux transporters: autoinhibited Ca 2+ -ATPases (ACAs) and Ca 2+ /H + exchangers (CAXs) [92,93]. Recent studies have demonstrated how phosphorylation serves as a mechanism for modulating the activities of these transporters, thereby orchestrating the termination of Ca 2+ signals and restoring cellular Ca 2+ homeostasis.

ACAs are P-type IIB ATPases that pump Ca 2+ from the cytosol into intracellular compartments like endoplasmic reticulum and vacuole, or extracellular space across the plasma membrane. These pumps are autoinhibited by their N-terminal regulatory domain, where a stalk region connects the transmembrane helices to the ATPase catalytic core, suppressing transport activity under the resting state [94]. Upon elevated cytosolic Ca 2+ levels, CaM binds to the N-terminal autoinhibitory domain of ACA, inducing a conformational change that relieves autoinhibition and activates the pump [95,96]. In addition to CaM binding, plant ACAs are regulated through phosphorylation at specific serine residues. In arabidopsis, ACA2 is phosphorylated by CDPK1 at a serine residue located downstream of the CaM-binding site within the autoinhibitory domain, resulting in the inhibition of both basal and CaM-stimulated activity without disrupting CaM binding [97]. By contrast, ACA8 is phosphorylated by CDPK1 and CDPK16 at distinct residues, where phosphorylation at specific sites can either inhibit or activate the pump activity by modulating the kinetics and affinity of CaM interaction [98]. Interestingly, ACA8 can also be phosphorylated by the CBL1-CIPK9 complex, serving as a potential feedback mechanism to fine-tune Ca 2+ signal termination during stress conditions such as mechanical wounding [99].

Plant CAX transporters are vital for Ca 2+ homeostasis, facilitating the sequestration of excess cytosolic Ca 2+ into vacuoles through Ca 2+ /H + exchange [93,100]. Their activity is precisely regulated by phosphorylation within the N-terminal autoinhibitory domain, allowing dynamic control of Ca 2+ distribution in response to cellular signals [101]. A recent study has shown that Ca 2+dependent CBL-CIPK complexes phosphorylate a conserved serine cluster in the N-terminal domain of CAX1/3, relieving autoinhibition and enhancing vacuolar Ca 2+ sequestration under high-Ca 2+ conditions to prevent cellular toxicity. Additionally, during plant immune responses, the PTI pathway, mediated by the FLS2-BAK1-BIK1/PBL1 complex, independently phosphorylates the same serine cluster, fine-tuning Ca 2+ signaling during defense activation. This dual regulatory system enables plants to precisely modulate cytosolic Ca 2+ levels, balancing growth and immune responses in the fluctuating environment [80].

Reversible phosphorylation of specific residues by kinases and phosphatases alters the physical and chemical properties of the target proteins, triggering conformational changes that affect their activity, localization, stability, or interactions with other cellular components. In the context of phospho-regulation of plant transport proteins, recent structural studies have provided insights into how phosphorylation modulates the activity of these transporters at the atomic level. In general, phosphorylation typically occurs in the cytosolic domains of the transporters to which kinases have easy access. Upon phosphorylation, these domains often interact with the catalytic domains of the enzymes (in the case of ATPases) or pore-forming domains of the channels, inducing allosteric changes to modulate the transport activity. Additionally, phosphorylationinduced structural changes can influence interactions with other regulatory proteins, either by creating new interaction interfaces or disrupting existing ones. Several examples are highlighted here to explore the mechanistic and structural aspects of phosphorylation-mediated regulation on plant transporters.

Phosphorylation can directly modulate the gating of ion channels by controlling their transition between open and closed states. Increased negative charges by adding a phosphate group to specific serine or threonine residue within a domain can trigger conformational changes that alter channel activity (Figure 3A). This dynamic modification enables ion channels to rapidly adjust ion flux in response to environmental and developmental signals. Upon phosphorylation of an autoinhibited ion channel, the channel undergoes a conformational change that enables interaction with the activator, leading to channel opening and ion transport. (D) Phosphorylation-regulated trafficking of ion channels. A channel initially retained in endosomal compartments undergoes phosphorylation, triggering its trafficking to the destined membrane for function. This regulatory mechanism controls the spatial distribution of channels to fine-tune ion homeostasis. In all panels, phosphorylation events are indicated by 'P' inside a purple circle. Ion species (A + /A -) are labeled and black arrows denote the direction of ion fl ow. A well-studied example is the K + channel AKT1, the activity of which strictly depends on phosphorylation by the CBL-CIPK complex. Electrophysiological studies showed that AKT1 remains inactive without kinase-mediated phosphorylation [27,102], highlighting phosphorylation as a direct gating mechanism. Structural studies have further revealed that phosphorylation-driven symmetry transitions in the cytoplasmic domains of AKT1 are key to the mechanisms regulating AKT1 channel activity. Phosphorylation at specific serine residues, such as Ser-26 and Ser-338, triggers a shift from a twofold to a fourfold symmetric arrangement in the cytoplasmic regions. This rearrangement removes structural restrictions, allowing parts of the protein to adopt a configuration that opens the channel [103]. Additionally, AKT1 forms a heteromeric complex with the K + channel subunit 1 (KC1), where KC1 serves as an inhibitory subunit that suppresses AKT1 activity [104]. The phosphorylation-induced structural reorganization in AKT1 may also help overcome this inhibition by altering the interaction between the two proteins, ultimately facilitating channel opening and K + influx [103,105].

Phosphorylation-dependent activation of SLAC1 further exemplifies how phosphorylation dynamically controls channel gating. SLAC1 remains in an autoinhibited state through interactions between its cytosolic regulatory domain (CRD) and transmembrane domain. Structural studies have identified conserved motifs in the N-and C-terminal cytosolic regions as key elements maintaining its closed conformation [106]. Phosphorylation at key serine (S) residues, such as S59, S86, and S120, releases the CRD 'plug' from the pore, initiating conformational changes that enable anion permeation [106,107]. Cryo-electron microscopy (EM) analyses revealed a two-phase activation mechanism: an inhibition-release phase, where the CRD disengages from the pore, and a subsequent binding-activation phase, in which phosphorylated residues interact with positively charged intracellular loops, leading to pore dilation [107]. Electrophysiological analyses and structure-function studies further demonstrated that phosphorylation-induced structural rearrangements in the pore helices and cytosolic loops are critical for precise regulation of anion efflux during guard cell signaling [108].

Phosphorylation can also modulate the gating of plant channels by integrating multiple signals. In two-pore channel 1 (TPC1), structural analyses have identified multiple phosphorylation sites across distinct subdomains that may allosterically influence the channel sensitivity to cytosolic Ca 2+ and voltage [109]. This multilayered regulation fine-tunes TPC1 activity to provide tight regulation of vacuolar ion flux.

Many transport proteins have autoinhibitory domains that keep them in inactive conformations under resting states. These autoinhibitory domains often work by binding to the transport domains, preventing conformational changes required for activity. Phosphorylation introduces negative charges and causes structural rearrangements to relieve autoinhibition and activate the protein (Figure 3B). For example, structural studies revealed that SOS1 exists as a homodimer and remains autoinhibited through interactions between its transmembrane and intracellular domains [110,111]. Phosphorylation by the CBL4/SOS3-CIPK24/SOS2 kinase complex targets specific residues in the C-terminal tail, disrupting the inhibitory interactions and inducing structural changes that convert SOS1 into an active, outward-facing state capable of Na + extrusion [111,112].

The arabidopsis Ca 2+ /H + exchangers CAX1 and CAX3 are both autoinhibited by their N-terminal regulatory domains, which interact with the transport core to keep the exchanger in an inactive state [101,113]. A dominant-negative mutation in the autoinhibitory domain of CAX1 further confirmed the regulatory function of this region [114]. Structural modeling and simulation suggest that this N-terminal domain may interact with the transmembrane core to stabilize an inactive state. In response to high-Ca 2+ stress or elevated cytosolic Ca 2+ levels, phosphorylation of a conserved serine-rich cluster in the N-terminal region induces conformational changes that relieve the autoinhibition, allowing activation of CAX transporters [80]. Interestingly, the yeast homolog VCX1 lacks such an N-terminal inhibitory domain and its overall structure supports a constitutively active mode of Ca 2+ transport [115]. The evolution of phosphorylation-dependent activation in plant CAX transporters allows plants to dynamically regulate Ca 2+ sequestration into the vacuole. This mechanism prevents unnecessary energy expenditure while enabling plants to better balance growth and stress responses.

Extending this concept to boron transport, the exporter BOR1 was recently shown to be autoinhibited by its C-terminal tail [116]. A cryo-EM structure revealed that the C-terminal segment folds back to restrict the conformational transitions required for substrate translocation, thereby maintaining BOR1 in an inactive state. Displacement or removal of this autoinhibitory tail activates boron efflux, providing a mechanistic basis for conditional control of boron transport.

Protein-protein interactions Phosphorylation-mediated activation of membrane transporters can also be achieved by modulating protein-protein interaction: either by promoting the binding of positive regulatory components (Figure 3C) or by disrupting interactions with negative regulators. A well-known example of this mechanism is plasma membrane H + -ATPase. Phosphorylation of a conserved threonine residue in its C-terminal autoinhibitory domain enables the binding of 14-3-3 proteins [15,117]. Structural studies have revealed that phosphorylation of the C-terminal tail of plant H + -ATPase initiates its binding to a conserved groove in 14-3-3 proteins, promoting the assembly of an H + -ATPase-14-3-3 hexametric complex [118,119]. This interaction induces conformational changes that stabilize the active state of H + -ATPase, relieving autoinhibition and thereby enhancing proton pumping to maintain pH gradient and drive secondary nutrient uptake. By contrast, arabidopsis CNGC2-CNGC4 is negatively regulated by CaM, which binds to the channel complex to suppress its activity. Although the structural basis of CaM-mediated inhibition of plant CNGC channels remains unknown, studies in animal systems reveal that CaM binding induces structural remodeling, forming a compact ternary complex that stabilizes the closed state of CNGCs [120,121]. In arabidopsis, phosphorylation of CNGC4 likely alters the conformation of CNGC2-CNGC4 complex, disrupting CaM binding and relieving its inhibition to facilitate Ca 2+ influx in response to cellular signals [81]. Taken together, these examples suggest that phosphorylation modulates protein-protein interactions to dynamically adjust transporter activity, enabling plant cells to rapidly adapt to both internal and external stimuli.

In addition to directly modulating their activities, phospho-regulation can control the subcellular trafficking and localization of transport proteins. Phosphorylation modulates the dynamic distribution of these membrane proteins by regulating endocytosis, exocytosis, and membrane retention, leading to precise spatial and temporal control of ion transport in response to environmental and physiological cues (Figure 3D). This regulation occurs because phosphorylation can potentially alter key motifs or interaction sites on the transporters, influencing their recognition by trafficking machinery such as clathrin adapters, vesicle coat proteins, or scaffolding complexes [122,123]. For instance, phosphorylation of specific serine residues in the C-terminal tail of PIP2;1 triggers endocytosis, reducing its abundance at the plasma membrane to limit water loss under salt stress. Conversely, reduced phosphorylation stabilizes PIP2;1 at the plasma membrane, promoting water uptake and maintaining cellular water homeostasis under normal conditions [124]. Another example is the PO 4 3-transporter (PHT; PT) family critical for PO 4 3-acquisition from the soil.

Under PO 4 3--sufficient conditions, phosphorylation of PTs promotes their internalization and subsequent degradation, limiting PO 4 3-uptake to prevent toxicity [125,126]. By contrast, during phosphate starvation, a protein phosphatase dephosphorylates PTs and stabilizes them at the plasma membrane, thereby enhancing PO 4 3-absorption to support plant growth and metabolism [127]. The polar distribution of PIN auxin transporters is also regulated by phosphorylation. The PINOID-mediated phosphorylation promotes the polar localization of PINs to specific plasma membrane domains, directing auxin flow and supporting developmental processes such as organogenesis and tropisms [128]. These phosphorylation-dependent trafficking mechanisms allow plants to dynamically control the localization and abundance of membrane transporters to adapt to fluctuating environmental conditions and coordinate growth and stress responses.

Autoinhibition represents a fundamental strategy to limit the activities of plant channels and carriers, conserving energy under 'resting state'. In response to changes in internal and external environments, cells shift to a 'signaling state' that typically involves phosphorylation to relieve autoinhibition and activate transporters, enabling the movement of ions, nutrients, and

What novel phospho-targets can be identified through large-scale phosphoproteomic studies to reveal new regulatory mechanisms in membrane transport?

How do different kinases coordinately regulate ion transporters under single and/or combined environmental stress conditions?

How do phospho-switches in ion transporters contribute to short-term responses versus long-term adaptation in plants?

What are the spatiotemporal dynamics of phosphorylation events in vivo, and how do they vary across distinct cellular compartments, cell types, and physiological contexts?

How does the integration of phosphorylation with other posttranslational modifications, such as ubiquitination, modulate the stability, localization, and activity of transport proteins?

What are the structural mechanisms through which phosphorylation activates specific ion channels and carriers?

How can phospho-regulation of ion transporters be harnessed to engineer crops with enhanced nutrient uptake, increased stress resilience, and improved agricultural performance? metabolites based on the new homeostatic status of the cell. The dynamic switch between inactive and active states allows plants to conserve energy while rapidly adjusting their transport activities to adapt to frequent environmental challenges during their life cycle. A key feature of phosphorylation-mediated regulation is its ability to integrate multiple signaling pathways. The same transporter can be phosphorylated by multiple kinases at the same or different sites, each representing a distinct signaling cascade and leading to a specific physiological response. This multilayered regulation allows transport proteins to act as versatile signaling hubs, coordinating diverse responses to environmental stresses, hormonal cues, and developmental signals.

Although significant progress has been made in understanding phosphorylation-mediated regulation of plant transport proteins, much remains unexplored (see Outstanding questions). Recent phospho-proteomic studies have revealed extensive phosphorylation sites on ion channels and carriers in plants under various conditions, providing a wealth of potential targets for future research exploring regulatory mechanisms governing nutrient uptake, ion homeostasis, and stress adaptation. To fully unravel the complexity of autoinhibition and phospho-regulation, further studies should integrate phospho-proteomics with genetics, structural biology, and real-time imaging. Major areas of focus include: (i) deciphering how phosphorylation interacts with other post-translational modifications, such as ubiquitination, to fine-tune transport activity; (ii) mapping the spatial and temporal dynamics of phosphorylation and autoinhibitory mechanisms in response to specific stimuli to understand how signals are integrated and converged to membrane transport; (iii) resolving high-resolution structures of transport proteins in distinct autoinhibited and phosphorylated states to uncover conformational transitions and activation mechanisms; and (iv) rationally designing transporters to perform specific functions under various conditions based on their structures and regulatory switches, which can potentially improve agronomic traits in crops. Advancing our knowledge in ion transport and signaling will pave the way for innovative strategies to enhance plant resilience and nutrient use efficiency, supporting sustainable agricultural practices to address the mounting challenges posed by climate change.