The plant hormone abscisic acid (ABA) serves as a physiological monitor to assess the water status of plants and, under drought conditions, induces stomatal pore closure by activating specific ion channels, such as a slow-anion channel (SLAC1) that, in turn, mediate ion efflux from the guard cells. Earlier genetic analyses uncovered a protein kinase (OST1) and several 2C-type phosphatases, as respective positive and negative regulators of ABAinduced stomatal closure. Here we show that the OST1 kinase interacts with the SLAC1 anion channel, leading to its activation via phosphorylation. PP2CA, one of the PP2C phosphatase family members acts in an opposing manner and inhibits the activity of SLAC1 by two mechanisms: (1) direct interaction with SLAC1 itself, and (2) physical interaction with OSTI leading to inhibition of the kinase independently of phosphatase activity. The results suggest that ABA signaling is mediated by a physical interaction chain consisting of several components, including a PP2C member, SnRK2-type kinase (OST1), and an ion channel, SLAC1, to regulate stomatal movements. The findings are in keeping with a paradigm in which a protein kinase-phosphatase pair interacts physically with a target protein to couple a signal with a specific response.anion transport ͉ protein dephosphorylation ͉ protein phosphorylation ͉ signal transduction A bscisic acid (ABA) has been found to act as a signal in modulating the activity of both K ϩ and anion channels of the plasma membrane of guard cells (1-3). Based on inhibitor studies, protein kinases and phosphatases constitute an interface between ABA and the ion channel (4-8). Several protein kinases and phosphatases that regulate ABA signaling have been identified in Arabidopsis with genetic approaches. For instance, ABI1 and ABI2 are closely related protein phosphatase 2C (PP2C) members that negatively regulate ABA response (9-11). In a later study, two other PP2C members related to ABI1 and ABI2 were identified in seed germination mutants showing a hypersensitive response to ABA (12). In another line of investigation, reverse genetic procedures have led to the identification of additional PP2C members including PP2CA, HAB1, and HAB2 that play a role in ABA signaling (13)(14)(15). Each of these ABA-signaling members belongs to the ''A type'' PP2C phosphatase family (16).An opposing participant, notably an ABA-activated protein kinase (AAPK), was uncovered early on with Vicia faba (17). In subsequent work, a mutant of the AAPK homologue was identified in Arabidopsis as Open Stomata mutant ost1 (18). The ost1 mutant is ABA-insensitive and keeps its stomata open, even under drought conditions. OST1 is a member of the Arabidopsis SnRK2-type protein kinase family that includes other members functional in the ABA response (19)(20)(21)(22). Each of these SnRK2 kinases is involved in the ABA response and is activated upon ABA treatment.Collectively, previous work has demonstrated that SnRK2-type kinases and PP2C-type phosphatases play a major role in ABA signaling, le...

Potassium (K ؉ ) is an essential nutrient for plant growth and development. Plants often adapt to low K ؉ conditions by increasing their K ؉ uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K ؉ transporter 1 (AKT1), responsible for K ؉ uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel.

Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)-A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.signal transduction | root growth | plant hormones

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO42−, NO3−, Cl−, and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.

Arabidopsis K+ transporter 1 (AKT1) participates in K+ uptake in roots, especially under low-K conditions. We recently identified a Ca²⁺ signaling pathway consisting of multiple calcineurin B-like calcium sensors (CBLs) and multiple target kinases (CBL-interacting protein kinases or CIPKs) that phosphorylate and activate AKT1, whereas a specific PP2C-type phosphatase inactivates CIPK-dependent AKT1 activity. In this study, we analyzed the interactions between PP2Cs and the CBL-CIPK pathway and found previously unsuspected mechanisms underlying the CBL-CIPK-PP2C signaling processes. The interaction between the CIPKs and PP2Cs involves the kinase domain of the CIPK component, in addition to the protein phosphatase interacting motif (PPI) in the regulatory domain. Furthermore, specific CBLs physically interact with and inactivate PP2C phosphatases to recover the CIPK-dependent AKT1 channel activity. These findings provide further insights into the signaling network consisting of CBL-CIPK-PP2C interactions in the activation of the AKT1 channel.

No abstract

Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neurons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code and decode Ca(2+) signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32, controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca(2+) accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activation of CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity. Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, similar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading to more severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in which calcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca(2+) oscillations in the polar growth of pollen tubes.

Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.