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Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00383-3
10.1016/j.xplc.2024.101042
101042
Commentary
Phosphorylation of CAX transporters controls Ca2+ homeostasis
Zhou Jinggeng jason@shnu.edu.cn
∗
He Yunxia
Meng Xiangzong xzmeng@shnu.edu.cn
∗∗
Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
∗ Corresponding author jason@shnu.edu.cn
∗∗ Corresponding author xzmeng@shnu.edu.cn
19 7 2024
09 9 2024
19 7 2024
5 9 10104221 5 2024
25 6 2024
17 7 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Published: July 19, 2024
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pmcMain text

Plants rely on a sophisticated innate immune system to recognize and defend against invading pathogens. The first line of plant innate immunity, called pattern-triggered immunity (PTI), is initiated upon detection of pathogen-associated molecular patterns (PAMPs) by plasma-membrane-localized pattern recognition receptors (PRRs) (Jones and Dangl, 2006). A well-characterized PRR is the Arabidopsis receptor-like kinase (RLK) FLS2, which complexes with the co-receptor BAK1 to detect the bacterial PAMP flagellin (or its major epitope flg22) (Zipfel, 2014). Downstream of PAMP-triggered PRR activation, receptor-like cytoplasmic kinases (RLCKs), such as BIK1 and RIPK, associate with and are activated by PRRs to trigger diverse downstream immune signaling events, including reactive oxygen species (ROS) burst, phosphatidic acid (PA) production, increased cytoplasmic calcium concentration ([Ca2+]cyt), and activation of mitogen-activated protein kinase cascades, which collectively lead to the establishment of plant PTI (Bigeard et al., 2015).

Ca2+ is a prominent second messenger, and the rapid and transient increase in [Ca2+]cyt during PTI is sensed by Ca2+ decoders, including calcium-dependent protein kinases (CDPKs), calmodulins (CaMs), and calcineurin B-like (CBL) proteins, to transduce immune signals (Yuan et al., 2017). Several plasma-membrane-localized ion channels that mediate Ca2+ influx into the cytosol during PTI, such as the cyclic nucleotide-gated channel proteins CNGC2/4, have been identified (Tian et al., 2019). Because excess [Ca2+]cyt is toxic to plants, it is essential to restore a low resting [Ca2+]cyt level after immune activation. However, the mechanisms by which excess cytoplasmic Ca2+ is scavenged after immune-stimulated increases in [Ca2+]cyt have long remained elusive.

Very recently, Wang et al. performed a genetic screen for Arabidopsis mutants with increased [Ca2+]cyt in response to high external Ca2+ and identified two vacuolar Ca2+/H+ exchangers, CAX1/3, that were essential for cytoplasmic Ca2+ efflux, preventing excessive [Ca2+]cyt in response to external Ca2+ or flg22 (Wang et al., 2024). In wild-type Arabidopsis expressing a Ca2+ biosensor (MatryoshCaMP6s), treatment with high external Ca2+ or flg22 rapidly induced an increase in [Ca2+]cyt, which was then restored to resting levels within minutes (Tian et al., 2019; Wang et al., 2024). By contrast, the cax1/3 single and double mutants failed to reset [Ca2+]cyt, leading to prolonged elevation of [Ca2+]cyt in response to external Ca2+ or flg22, indicating that the CAX1/3 transporters are involved in mediating cytoplasmic Ca2+ efflux. Notably, [Ca2+]cyt was higher in the cax1/3 double mutant than in the cax1 single mutant, indicating that CAX1/3 function redundantly in mediating cytoplasmic Ca2+ efflux. This redundancy was also supported by the observation that cax1/3 displayed stunted growth, whereas the single mutants grew normally (Wang et al., 2024).

In exploring how CAX1/3 are activated by external Ca2+ or flg22, Wang et al. hypothesized that the N-terminal autoinhibitory domains of CAX1/3, which face the cytosol, may be involved in their activation (Mei et al., 2007; Wang et al., 2024). Alignment of the CAX autoinhibitory domain sequences revealed a cluster of four consecutive serine residues (referred to as an S-cluster) that was strictly conserved in angiosperm CAX1/3 orthologs and was frequently phosphorylated in phosphoproteomic analyses, leading the authors to speculate that phosphorylation of the S-cluster may be involved in CAX1/3 activation at elevated [Ca2+]cyt. In a search for potential protein kinases targeting CAX1/3, three CBL-interacting protein kinases (CIPK3/9/26) and two CBL Ca2+ sensors (CBL2/3) were identified as candidate regulators of CAX1/3 because of their interaction in the tonoplast where CAX1/3 reside and their co-expression with CAX1/3 in roots and leaves. Importantly, all CBL–CIPK pairs among CBL2/3 and CIPK3/9/26 could activate CAX1/3 transporters when co-expressed in yeast cells in a complementation assay. Furthermore, compared with wild-type plants, the cbl2 cbl3, cipk3 cipk9 cipk26, and cax1 cax3 mutants all showed similar stunted growth phenotypes in soil and displayed sustained [Ca2+]cyt elevation in response to high external Ca2+ (Wang et al., 2024). These results suggested that CBL2/3–CIPK3/9/26 modules may directly phosphorylate, and thereby activate, CAX1/3. Indeed, in vitro phosphorylation assays showed that CAX1/3 could be phosphorylated by CIPK3/9/26 in a CBL2/3-dependent manner. Mass spectrometry analysis and functional identification demonstrated that CBL2/3–CIPK3/9/26 modules phosphorylate the conserved S-clusters in the autoinhibitory domains of CAX1/3 to activate them. Because CBLs are Ca2+ sensors, Wang et al. hypothesized that a CBL–CIPK–CAX cascade may mediate the signaling from perception of [Ca2+]cyt to sequestration of excessive Ca2+ in the vacuole. Indeed, a CBL3 mutant that lost Ca2+-binding ability failed to enhance the CIPK9-mediated phosphorylation of CAX1/3 and thus hindered CBL–CIPK-mediated CAX1/3 activation in yeast and Arabidopsis (Wang et al., 2024). These results indicate that the CBL–CIPK–CAX cascade links Ca2+ perception to phosphoactivation of CAX1/3 for removal of excess cytoplasmic Ca2+.

To investigate whether flg22 also induced the phosphoactivation of CAX1/3 transporters to reset [Ca2+]cyt, Wang et al. tested the phosphorylation status of CAX1/3 in Arabidopsis mesophyll protoplasts during flg22 elicitation. Indeed, rapid phosphorylation of CAX1/3, but not other CAX members, was observed in response to flg22 treatment. The subsequent observation that disruption of the CBL–CIPK pathway did not affect flg22-induced CAX1/3 phosphorylation implied the existence of other flg22-responsive kinase(s) that phosphorylate CAX1/3. In search of such kinase(s), Wang et al. tested the involvement of key kinases downstream of the LRR receptor FLS2 in the flg22 pathway and found that the RLK co-receptor BAK1 and the RLCKs BIK1/PBS1-like kinase 1 (PBL1) were required for flg22-induced CAX1/3 phosphorylation. Furthermore, both in vitro and in vivo phosphorylation assays demonstrated that BIK1/PBL1 phosphorylated the N-terminal autoinhibitory domains of CAX1 and CAX3 (Wang et al., 2024). Interestingly, mapping of the BIK1/PBL1-mediated phosphorylation sites of CAX1/3 revealed that the serine residues phosphorylated by CIPK3/9/26 in the CAX1/3 S-cluster were also the major sites phosphorylated by BIK1/PBL1. Mutations of the S-cluster in the CAX1/3 autoinhibitory domain hindered flg22-induced and BIK1/PBL1-dependent phosphorylation of CAX1/3 (Wang et al., 2024). Given that CIPK3/9/26-mediated phosphorylation of the S-cluster in CAX1/3 led to their activation, these results indicate that BIK1/PBL1 also phosphorylate the S-cluster to activate CAX1/3 in response to flg22 and further suggest that the RLK–RLCK–CAX module links flg22 perception to phosphoactivation of CAX for resetting of [Ca2+]cyt.

As a central convergent regulator in plant immunity, BIK1 also phosphorylates and activates CNGC2/4 channels to mediate the PAMP-triggered Ca2+ influx, as reported by Tian et al. (2019). To discern the role of the BIK1/PBL1–CAX1/3 module in the regulation of Ca2+ efflux upon flg22 elicitation, Wang et al. designed a procedure in which pretreatment with high external Ca2+ to saturate Ca2+ influx was followed by flg22 treatment to specifically monitor the flg22-triggered Ca2+ efflux phase. They found that the flg22-triggered Ca2+ efflux phase was significantly reduced in both bik1 pbl1 and cax1 cax3 mutants. In addition, in bacteria-infiltrated leaves, both bik1 pbl1 and cax1 cax3 mutants showed higher [Ca2+]cyt, further supporting a role for the BIK1/PBL1–CAX1/3 module in mediation of excess cytoplasmic Ca2+ removal during pathogen invasion (Wang et al., 2024). Together, the recent study by Wang et al. and the earlier report by Tian et al. indicate that BIK1/PBL1 play dual roles in regulating Ca2+ influx and efflux to shape Ca2+ signaling during pathogen invasion.

Taken together, the findings of Wang et al. reveal two mechanisms by which CAX1/3 transporters are activated to enable the removal of excess cytoplasmic Ca2+ (Figure 1). Under normal conditions, the Ca2+–CBL–CIPK–CAX cascade monitors [Ca2+]cyt and sequesters excess Ca2+ in the vacuole to maintain [Ca2+]cyt at a resting level. During pathogen invasion, vacuolar Ca2+ sequestration activated by the RLK–RLCK–CAX module prevents a sustained increase in [Ca2+]cyt after immune activation. Unlike the Ca2+–CBL–CIPK–CAX cascade that undergoes Ca2+-dependent feedback regulation of cytoplasmic Ca2+ removal, the RLK–RLCK–CAX module represents a Ca2+-independent strategy for scavenging of cytoplasmic Ca2+ (Wang et al., 2024). Furthermore, several previous reports have characterized additional regulatory mechanisms of CAX1/3 activation, such as formation of a “hetero-CAX” complex and mediation by CAX-interacting proteins (CXIPs), including CXIP4 and the Ser/Thr kinase SOS2 (Bose et al., 2011). The multiple regulatory mechanisms that act on CAX1/3 provide substantial flexibility for the dynamic modulation of [Ca2+]cyt oscillations. Notably, the regulatory roles of these CXIPs were mainly explored by co-expression of CXIPs with CAXs in yeast two decades ago; we still lack in planta genetic evidence to support their involvement in the regulation of CAX1/3-mediated [Ca2+]cyt efflux. However, in a recent report by Wang et al., SOS2 (also referred to as CIPK24) was shown to be incapable of activating CAX1, and sos2 mutant and wild-type Arabidopsis plants exhibited similar growth in response to external Ca2+. Thus, the roles of these CXIPs in regulating CAX1/3-mediated efflux of cytoplasmic Ca2+ require further in planta investigation, as done by Wang et al. (2024). Once these regulatory pathways have been confirmed, a future challenge will be to explore how they are integrated and coordinated to activate CAX1/3 under specific conditions.Figure 1 A model depicting plant responses to external Ca2+ and bacterial invasion.

(A) Under normal conditions, external Ca2+ enters the cytoplasm through plasma-membrane-localized CNGC2/4 or other channels and transiently increases [Ca2+]cyt. This increase is sensed by the Ca2+ sensors CBL2/3, which recruit CIPK3/9/26 to the tonoplast and activate these kinases. CBL2/3-paired CIPK3/9/26 phosphorylate and activate CAX1/3 to transport excess cytoplasmic Ca2+ into the vacuole.

(B) During bacterial invasion, the plant PRR complex FLS2–BAK1 recognizes flagellin or its epitope flg22 and activates BIK1/PBL1, which in turn phosphorylate and activate CNGC2/4 channels (Ca2+ influx) and CAX1/3 transporters (Ca2+ efflux). The dual roles of BIK1 in the enhancement of Ca2+ influx and Ca2+ efflux shape the [Ca2+]cyt signal to generate the appropriate magnitude and persistence of immune responses. In addition to CAX1/3, tonoplast-localized ACA4/11, endoplasmic-reticulum-localized ACA1/2/7, and plasma-membrane-localized ACA8/10 also contribute to the removal of excess cytoplasmic Ca2+. CIPK- and CDPK-mediated phosphorylation of ACAs may fine-tune ACA pump activity.

In addition to the Ca2+/H+ exchangers CAX1/3, calcium-ATPase (ACA) pumps are also involved in the maintenance of [Ca2+]cyt homeostasis. Knockout mutants of tonoplast-localized ACA4/11 or endoplasmic-reticulum-localized ACA1/2/7 exhibited elevated basal [Ca2+]cyt and prolonged [Ca2+]cyt elevation during flg22 treatment (Hilleary et al., 2020; Rahmati Ishka et al., 2021), and knockout mutation of the plasma-membrane-localized ACA8/10 also resulted in increased [Ca2+]cyt levels, accompanied by autoimmune phenotypes (Yang et al., 2017). These ACAs thus appear to be involved in the delivery of cytoplasmic Ca2+ to the vacuole, endoplasmic reticulum, or apoplast for maintenance of [Ca2+]cyt homeostasis (Figure 1). How ACAs coordinate with CAXs spatially and temporally to regulate [Ca2+]cyt oscillations and thus plant stress responses is unknown. CAXs are low-affinity (Km = 10–15 μM) but high-capacity transporters, whereas ACAs are high-affinity (Km = 0.2–1 μM) but low-capacity transporters (Bose et al., 2011), suggesting that CAXs and ACAs may function differently in the removal of excess cytoplasmic Ca2+. CAXs may be predominantly involved in the removal of cytoplasmic Ca2+ when [Ca2+]cyt reaches a high threshold level under stress conditions, whereas ACAs may be mainly involved in fine-tuning of [Ca2+]cyt under normal and stress conditions. Future studies are needed to determine how CAXs act together with ACAs to maintain [Ca2+]cyt homeostasis.

Interestingly, all these CAX and ACA transporters contain an N-terminal autoinhibitory domain for regulation of their activities (Hilleary et al., 2020). The N-terminal autoinhibitory domain of ACA8 could bind to Ca2+-loaded CaMs to release its autoinhibition (Tidow et al., 2012), and phosphorylation of the N-terminal autoinhibitory domain of ACA8 mediated by the Ca2+–CBL1–CIPK9/14 module increased its pump activity (Costa et al., 2017). Conversely, CDPK1-mediated phosphorylation of ACA2 and CDPK1/16-mediated phosphorylation of ACA8 inhibited their pump activity (Hwang et al., 2000; Giacometti et al., 2012). Therefore, these findings, together with those of Wang et al. on CIPK3/9/26- and BIK1/PBL1-mediated phosphoregulation of CAX1/3, suggest that phosphorylation of the N-terminal autoinhibitory domain is likely a common regulatory mechanism of CAX and ACA transporters (Figure 1). It should be noted that the phosphoregulation of ACAs has been studied mainly in yeast. To support these conclusions, it will be necessary to obtain in planta evidence as done in the genetic analysis of CAXs by Wang et al. (2024). Once confirmed, it will be interesting to investigate how CAX and ACA phosphorylations by different upstream protein kinases are integrated and coordinated to regulate [Ca2+]cyt oscillations and homeostasis.

In addition to Ca2+, PA and ROS also act as important second messengers in plant immunity. During pathogen invasion, PA is generated primarily by the phosphorylation of diacylglycerol mediated by diacylglycerol kinase 5, and ROS production is catalyzed predominantly by respiratory burst oxidase homolog D. A recent report revealed that phosphorylation of diacylglycerol kinase 5 by BIK1 or MPK4 activated or attenuated the PA burst, respectively (Kong et al., 2024). Similarly, respiratory burst oxidase homolog D also showed opposite regulation when it was phosphorylated by BIK1 or PBL13 (an RLCK member) (Lee et al., 2020). Thus, phosphorylation appears to be a double-edged sword in the control of PA and ROS production. As for [Ca2+]cyt oscillations in plant immunity, in contrast to phosphoactivation of CAX-mediated vacuolar Ca2+ sequestration, phosphorylation-mediated attenuation of CAX activity has not been demonstrated and requires further investigation.

Funding

This work was supported by the National Natural Science Foundation of China (grants 32370291 to J.Z. and 32370313 to X.M.) and the Natural Science Foundation of Shanghai (grant 22ZR1446300 to J.Z.).

Acknowledgments

No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.
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