A BRI1–CNGC12 phosphorylation module links hormone signaling to manganese homeostasis in plants

Significance

Manganese (Mn) toxicity represents a significant constraint on plant growth in acidic soils. Calcium ions (Ca²⁺) function as critical second messengers in plants. While high-Mn stress is known to trigger Ca²⁺ signaling events that regulate Mn homeostasis, the mechanism generating the specific Ca²⁺ signals remains undefined. Here, we demonstrate that the cyclic nucleotide-gated channel CNGC12 is essential for generating Ca²⁺ oscillations within a specific “high-Mn-sensing niche” in Arabidopsis roots and for conferring Mn tolerance. Furthermore, we identify the brassinosteroid (BR) receptor BRI1 as a key regulator of CNGC12 channel activity. Mechanistically, phosphorylation of Ser22 within CNGC12 by BRI1 governs Ca²⁺ transport activity. Collectively, our findings delineate a BR–BRI1–CNGC12–Ca²⁺ signaling module as a core mechanism underpinning plant resilience to Mn toxicity.

Abstract

Manganese (Mn) toxicity in acidic or waterlogged soils severely impacts crop productivity. Although high-Mn stress triggers Ca²⁺ signals that regulate Mn homeostasis, the mechanism generating these signals remains unclear. Here, we show that the cyclic nucleotide-gated channel CNGC11/12 are essential for Mn tolerance, as cngc11/12 mutants exhibited hypersensitivity to Mn and cngc12 mutant showed reduced Ca²⁺ elevations. The brassinosteroid (BR) receptor BRI1 physically interacted with CNGC12 and phosphorylated Ser22 residue, a modification critical for channel activation. Accordingly, bri1 mutants displayed impaired Mn-induced Ca²⁺ signaling and heightened Mn sensitivity. Mn stress rapidly activated BRI1 kinase, peaking within minutes, and electrophysiological assays confirmed that BRI1-mediated phosphorylation gates CNGC12-dependent Ca²⁺ currents. Exogenous brassinolide treatment augmented high-Mn-induced Ca²⁺ signaling, BRI1-mediated CNGC12 phosphorylation, and high-Mn tolerance. Mutations in either BRI1 or CNGC12 abolished CPK5-dependent phosphorylation of MTP8 and impaired NRAMP1 endocytosis. Our study identifies the BRI1–CNGC12 module as a key node linking BR signaling to Ca²⁺-dependent Mn detoxification, revealing how phytohormone pathways regulate ion stress adaptation.

Manganese (Mn) is an important trace element for plants (1). However, excessive Mn accumulation in plants induces toxicity by disrupting photosynthesis, impairing enzymatic functions, and competitively inhibiting phosphorus/iron/magnesium uptake, manifesting as chlorosis, necrotic leaf spots, and growth retardation that reduce crop yields (2). Acidic and waterlogged soils markedly enhance Mn²⁺ availability, exacerbating Mn uptake and toxicity (3). Notably, acidic soils account for approximately 30% of global arable land, and the intensification of soil acidification due to excessive nitrogen fertilizer application and acid rain deposition has exacerbated Mn toxicity issues (4). Therefore, elucidating the molecular mechanisms governing Mn uptake and transport is critical for developing crop varieties resilient to acid-affected soils.

The calcium signaling cascade plays a key role in modulating plant tolerance to high-Mn stress. As a pivotal secondary messenger, Ca²⁺ generates spatiotemporal signatures upon environmental stress, decoded by calcium sensors to trigger downstream regulatory cascades (5, 6). Under excess Mn stress, the elicited specific Ca²⁺ signature activates the calcineurin-B-like (CBL)1/9-CBL-interacting protein kinase CIPK23 complex. This sensor-kinase module phosphorylates the plasma membrane (PM)-localized Mn transporter NRAMP1 (7, 8). This phosphorylation promotes clathrin-mediated endocytosis of NRAMP1, thereby restricting cellular Mn uptake and ultimately alleviating Mn toxicity (7, 9). Meanwhile, the Ca²⁺-dependent protein kinases CPK4/5/6/11 are activated to phosphorylate the vacuolar Mn transporter MTP8, which enhances its transport activity for sequestering excess Mn into vacuoles, thereby improving plant tolerance to Mn toxicity (10). During late stress phases, a distinct Ca²⁺ signal recruits the CBL2/3-CIPK3/9/26 complex to phosphorylate and inhibit MTP8 activity. This antagonistic regulation between CPK4/5/6/11 and CBL2/3-CIPK3/9/26 fine-tunes Mn compartmentalization, balancing stress adaptation with growth demand (11). However, the molecular mechanisms underlying the generation of the specific Ca²⁺ signatures under both Mn deficiency and Mn-toxicity conditions remain poorly understood.

Cyclic nucleotide-gated channels (CNGCs) act as Ca²⁺-permeable channels in plants that regulate multiple biological functions (12). CNGC16, a calcium channel, is crucial for stress tolerance in pollen reproductive development (13). CNGC18 is an important Ca²⁺ channel that guides the extension of pollen tubes to the ovule (14, 15). CNGC6, CNGC9, and CNGC14 synergistically regulate calcium oscillations required for the polar growth of plant root hairs (16). CNGC5/6/9/12, ABA activated calcium channels, function for ABA-induced stomatal closure in Arabidopsis (17). External nitrate application disrupts the interaction between calcium channel CNGC15 and nitrate transporter NRT1.1, leading to dissociation of the preexisting CNGC15-NRT1.1 complex on the PM. This dissociation restores CNGC15-mediated Ca²⁺ influx, which activates calcium-dependent protein kinases CPK10/30/ 32. Subsequently, these kinases phosphorylate the transcription factor NLP7 (NIN-LIKE PROTEIN 7), triggering its activation and downstream nitrate-responsive gene expression (18). In plant immunity, CNGC2/4 and CNGC19/20 mediate pathogen-induced Ca²⁺ signaling (19, 20). In rice, OsCNGC9, OsCNGC14, and OsCNGC16 mediate thermo-tolerance by regulating cold/heat-induced calcium signaling (21, 22). However, it is unclear whether CNGCs are also involved in regulating the Ca²⁺ signaling response to Mn stress in Arabidopsis. In addition, receptor-like kinases (RLKs) act as critical regulators of calcium channels, especially modulating CNGCs. CNGC2/4 are phosphorylated by BIK1 upon MAMP perception, elevating cytosolic Ca²⁺ (19). During early cold stress, PSY1R phosphorylates CNGC20 to enhance Ca²⁺ transport, while CRPK1 promotes its degradation in later stages (23). In plant immunity, BAK1/SERK4 phosphorylates CNGC20, triggering proteasomal degradation rather than channel activation (20).

Brassinosteroids (BRs), the sixth major class of plant hormones, play important roles in plant growth, development, and stress response (24). BR bind to the receptor BR Insensitive 1 (BRI1), a leucine-rich receptor kinase located on the PM (25), BRI1 forms heterodimers with coreceptor BRI1-Associated Receptor Kinase 1 (BAK1), while mutual phosphorylation occurs (26, 27). The formation of BRI1–BAK1 heterodimers triggers sequential phosphorylation cascades via BR Signaling Kinase 1 (BSK1)/Constitutive Differential Growth 1 (CDG1) to activate BRI1-SUPPRESSOR 1 (BSU1) phosphatase (28). BSU1 dephosphorylates BR Insensitive 2 (BIN2) and inactivates it (28), coupled with Protein Phosphatase 2A (PP2A)-dependent activation of Brassinazole Resistant 1 (BZR1)/bri1-EMS Suppressor 1 (BES1) transcription factors that translocate to the nucleus and bind target gene promoters (29–31). BR signaling pathway is involved in various plant growth and development processes, as well as biological and abiotic stress responses, including cell elongation and division, leaf growth, pollen development, seed development, stomatal formation, flowering, and stress tolerance (32–36). Given the roles of BR signaling in multiple biological processes—including development and stress responses—we hypothesized its participation in Mn homeostasis regulation.

In this study, we demonstrate that the Ca²⁺ channel CNGC12 mediates high-Mn-induced cytosolic Ca²⁺ influx. The BR receptor BRI1 phosphorylates CNGC12 to enhance its channel activity, thereby activating Ca²⁺ sensors that phosphorylate MTP8 and NRAMP1. This promotes vacuolar Mn²⁺ sequestration via MTP8 and reduces NRAMP1-mediated Mn uptake through endocytosis, collectively contributing to the Mn toxicity tolerance. Collectively, our study elucidates the molecular mechanism by which the BRI1–CNGC12 module positively regulates high Mn stress-induced calcium signaling to resist high-Mn toxicity. Furthermore, we demonstrate that BRI1 serves as a convergence node bridging Ca²⁺ signaling and BR signaling pathways to coordinate Mn detoxification.

Results

The Calcium Channel CNGC12 Is Essential for High-Mn Stress Tolerance.

Emerging evidence indicates that high-Mn stress activates Ca²⁺ signaling cascades, which orchestrate Mn²⁺ homeostasis through phosphorylation-dependent modulation of Mn transporters MTP8/NRAMP1 activity mediated by Ca²⁺-sensors CPKs and CBL–CIPK complexes (6). CNGC can rapidly mediate the influx of Ca²⁺ into the cytoplasm, triggering the crucial calcium signaling cascade (12). To investigate the contribution of Ca²⁺ channels to high-Mn-induced Ca²⁺ influx, we collected a set of T-DNA insertion mutants in genes encoding CNGCs, and tested their high-Mn stress tolerance. Among all the CNGC mutants, loss-of-function alleles of cngc11 and cngc12 exhibit hypersensitive phenotypes to high-Mn stress, characterized by shorter root length and chlorosis, with cngc12 being the most sensitive (Fig. 1A). However, under normal conditions, no significant phenotypic differences were observed between the cngc mutants and WT plants (Fig. 1A). To assess functional redundancy between CNGC11 and CNGC12, we generated two cngc11/12 double mutant alleles by disrupting CNGC11 via CRISPR-Cas9 in the cngc12 mutant background (SI Appendix, Fig. S1). The cngc11/12 double mutant displayed a more pronounced high-Mn sensitive phenotype than either single mutant (Fig. 1 B and C), demonstrating that CNGC11 and CNGC12 function redundantly to positively regulate the response to high-Mn stress in Arabidopsis.

Fig. 1.

CNGC11/12 enhanced high-Mn tolerance in Arabidopsis. (A) Primary root lengths of WT Col-0 and cngcs mutant under high-Mn stress. Four-day-old seedlings were transferred onto 1/2 MS medium supplemented with or without 1.5 mM MnCl₂ (n = 15 seedlings). (B) Mn toxicity phenotypes of WT, cngc11, cngc12, and cngc11/12 mutants. The plants were grown and stressed as in A. (C) Statistical analysis of primary root lengths of plants shown in B (n = 15 seedlings). (D) Statistical analysis of Chla concentrations in plants shown in B (n = 15 seedlings). (E) Statistical analysis of Chlb concentrations in plants shown in B (n = 15 seedlings). (F) Statistical analysis of Mn concentrations in plants shown in B (n = 3 biological replicates). All data in this figure are presented as mean ± SD (n = 45 seedlings from three biologically independent experiments). Asterisks indicate significant differences (*P < 0.05, **P < 0.01; two-tailed Student’s t test).

Mn toxicity is known to cause leaf chlorosis (4) and leads to excessive Mn accumulation in plants (1). Therefore, we quantified chlorophyll and Mn concentrations in cngc11, cngc12, cngc11/12 mutants, and WT plants. Under nontoxic Mn conditions, no significant differences were detected in chlorophyll or Mn concentrations (Fig. 1 D–F). However, when plants were subjected to 1.5 mM MnCl₂, the concentrations of Chla and Chlb were significantly reduced in the cngc11, cngc12, and cngc11/12 mutants compared to the WT (Fig. 1 D and E). Consistent with increased sensitivity, the Mn concentration was significantly elevated in the cngc11, cngc12, and cngc11/12 mutants (Fig. 1F). Taken together, these results demonstrate that under high-Mn stress, the cngc11, cngc12, and cngc11/12 mutants accumulate less chlorophyll and more Mn than the WT. This indicates that CNGC11 and CNGC12 are involved in high-Mn detoxification and the regulation of Mn homeostasis.

BRI1 and BR Signaling Promote Plant Tolerance to High-Mn Stress.

RLKs are key regulators of calcium channels (37). To investigate the regulatory mechanisms of CNGC12, we systematically screened the phenotypes of the rlk mutant collections under high-Mn stress. This approach identified two allelic mutants of BRI1 (bri1-301 and bri1-5) that displayed pronounced hypersensitivity to Mn toxicity (SI Appendix, Fig. S2 and Fig. 2 A–C). The two mutants exhibit distinct molecular lesions with differential impacts on BRI1 function: The bri1-5 mutant harbors a point mutation causing protein misfolding and endoplasmic reticulum retention, thereby disrupting BR signaling (38), while bri1-301 carries a kinase-domain mutation that abolishes catalytic activity while maintaining protein stability (39). Under normal growth conditions, the bri1-301 mutant showed no significant difference in posttransplant root elongation compared to WT. Under high-Mn stress conditions, the bri1-301 mutant exhibited significantly more sensitivity phenotype than the WT plants, manifested by its severely inhibited primary root elongation and visible leaf chlorosis (Fig. 2A and SI Appendix, Fig. S3A).

Fig. 2.

BRI1 positively regulates plant high-Mn stress tolerance. (A) Statistical analysis of primary root lengths of WT (Col-0) and bri1-301 mutant under high-Mn stress (n = 15 seedlings). Four-day-old seedlings were transferred onto 1/2 MS medium supplemented with or without 1.5 mM MnCl₂. (B) Statistical analysis of primary root lengths of plants shown in WT (Ws2) and bri1-5 mutant under high-Mn stress (n = 15 seedlings). The plants were grown and stressed as in A. (C) Statistical analysis of the ratio of root length under high Mn stress treatment to that under normal conditions of plants in B. (D) Statistical analysis of primary root lengths of plants shown in WT (Col-0) and bak1-4, bak1-3, serk4-1, bak1-3 serk4-1 mutants under high-Mn stress (n = 15 seedlings). The plants were grown and stressed as in A. (E) Statistical analysis of the ratio of root length under high Mn stress treatment to that under normal conditions of plants in D. (F) Statistical analysis of primary root lengths of plants shown in WT (Col-0) and bes1-D, bzr1-1D mutants under high-Mn stress (n = 15 seedlings). The plants were grown and stressed as in A. (G) Statistical analysis of primary root lengths of plants shown in WT (Ws2) and bin2-3 bil1 bil2 mutants under high-Mn stress (n = 15 seedlings). The plants were grown and stressed as in A. (H) Statistical analysis of root lengths of WT plants under eBL- and BRZ- treated conditions. WT plants were grown for 4 d under normal conditions and transferred to fresh 1/2 MS medium with/without 1 nM eBL or 100 nM BRZ, or high-Mn stress medium (1/2 MS containing 1.5 mM MnCl₂ with/without 1 nM eBL or 100 nM BRZ). (I) Statistical analysis of the root length ratio (high-Mn stress/normal conditions) in H. All data in this figure are presented as mean ± SD (n = 45 seedlings from three biologically independent experiments). Asterisks indicate significant differences (**P < 0.01; two-tailed Student’s t test).

In addition, the bri1-5 mutant also exhibited a high-Mn sensitive phenotype. Under control conditions, its primary root elongation was significantly reduced vs. WT, a phenotype exacerbated under high-Mn stress with further suppression of root growth. We further quantified the relative root elongation rates of Mn-stressed vs. unstressed plants in both bri1-5 mutants and WT. Comparative analysis revealed significantly attenuated root elongation ratios in bri1-5 relative to WT (Fig. 2 B and C and SI Appendix, Fig. S3B), indicating that the bri1-5 mutant also exhibited sensitivity to high-Mn toxicity. Furthermore, chlorophyll and Mn concentrations were quantified in bri1-301, bri1-5 mutants, and WT. Under nontoxic Mn conditions, no significant differences were detected in Mn concentrations, whereas the chlorophyll content in both bri1-301 and bri1-5 mutants was significantly higher than in WT plants (SI Appendix, Fig. S3 G, H, J, and K). When exposed to high-Mn stress, both bri1-301 and bri1-5 mutants exhibited significantly reduced chlorophyll levels and elevated Mn accumulation relative to the WT (SI Appendix, Fig. S3 M and N). The results indicate that BRI1 plays a positive regulatory role in the plant tolerance to high-Mn stress.

To investigate whether CNGC12 and BRI1 regulate plant responses to Mn deficiency, we analyzed the phenotypes of cngc12 and bri1-301 mutants. Neither mutant exhibited manganese deficiency-specific phenotypes. Under both Mn-sufficient and deficient conditions, root lengths of cngc12 mutant did not differ significantly from wild-type (WT) plants. In addition, bri1-301 mutant displayed constitutively reduced root growth compared to wild type under all conditions, but the root length ratio (Mn-deficient/sufficient conditions) showed no significant difference between bri1-301 and WT plants (SI Appendix, Fig. S4). These results confirmed that BRI1 acts on CNGC12 to specifically regulate plant tolerance to high-Mn stress.

As BRI1 is the receptor for BR, we investigated whether BR signaling contributes to plant adaptation to high-Mn stress. We therefore analyzed high-Mn responses in mutants of two SERK family members: BAK1 (BRI1-Associated Receptor Kinase 1) and SERK4 (Somatic Embryogenesis Receptor-like Kinase 4). Under high-Mn conditions, single mutants bak1-3, bak1-4, and serk4-1 exhibited no significant phenotypic differences compared to the wild type (WT). In contrast, the bak1-3 serk4-1 double mutant displayed pronounced Mn hypersensitivity, manifesting severely suppressed root elongation (Fig. 2 D and E and SI Appendix, Fig. S3).

BES1 and BZR1, core transcription factors regulating BR-responsive gene expression, are inactivated by BIN2-mediated phosphorylation, leading to cytoplasmic retention and proteasomal degradation. To further probe BR signaling involvement, we examined the gain-of-function alleles bes1-D and bzr1-1D, as well as the bin2-3 bil1 bil2 triple mutant. All three genotypes conferred enhanced tolerance to high-Mn stress (Fig. 2 F and G and SI Appendix, Fig. S3).

Consistent with genetic evidence, pharmacological treatments modulated Mn tolerance: Application of the BR analog 24-epibrassinolide (eBL) enhanced Arabidopsis tolerance to high Mn, whereas the BR biosynthesis inhibitor brassinazole (BRZ) reduced tolerance (Fig. 2 H and I and SI Appendix, Fig. S3). In summary, BR signaling pathway confers high-Mn stress tolerance in Arabidopsis.

BRI1-Mediated High-Mn Stress Tolerance Requires CNGC12.

To explore the genetic relationship between BRI1 and CNGC12, we generated BRI1-OE/cngc12 plants by crossing BRI1-overexpressing (BRI1-OE) transgenic lines with the cngc12 mutant. The results demonstrated that BRI1-OE transgenic plants exhibited a high-Mn tolerant phenotype, whereas the BRI1-OE/cngc12 plants, similar to the cngc12 mutant, displayed a high-Mn stress-sensitive phenotype (SI Appendix, Fig. S5). These results reveal the epistasis of CNGC12 over BRI1, highlighting that BRI1 act upstream of CNGC12. We also checked for coexpression of BRI1 and CNGC12, as this would be a prerequisite for their potential interaction. Previous research demonstrates that both proteins exhibit consistent PM localization (40, 41). In this study, confocal microscopy analysis of ProCNGC12:CNGC12-GFP and ProBRI1:BRI1-GFP transgenic lines confirmed root tip expression and PM localization for both proteins, with unaltered subcellular distributions following high-Mn²⁺ stress (SI Appendix, Fig. S6). These results indicate the coexpression and colocalization of BRI1 and CNGC12, suggesting potential common function in modulating Ca²⁺ influx dynamics during high-Mn stress adaptation.

BRI1–CNGC12 Positively Regulates High-Mn-Induced Ca²⁺ Influx.

Since CNGC12 functions as a Ca²⁺-permeable channel, we investigated whether CNGC12 contributes to high-Mn induced increases in [Ca²⁺]_cyt. To this end, we generated WT, cngc12 seedlings expressing a construct encoding the Ca²⁺-binding fluorescent protein GCaMP6s-mCherry. Upon Ca²⁺ binding, GCaMP6s exhibits enhanced GFP fluorescence, while mCherry serves as an invariant reference—enabling ratiometric imaging of [Ca²⁺]_cyt via the GFP/mCherry signal ratio. We exposed these seedlings to a high-Mn stress and measured GFP/mCherry signal. The cngc12 seedlings showed a decrease in high-Mn induced GFP signals compared to WT seedlings, whereas we observed no significant difference between WT and cngc12 before high-Mn treatment (Fig. 3 A and B). Under high-Mn stress, the cngc12 mutant exhibited a diminished increase in [Ca²⁺]_cyt after high-Mn compared to WT (Fig. 3 A and B). Together, these results indicate that CNGC12 participates in high-Mn-evoked increases in [Ca²⁺]_cyt in plants.

Fig. 3.

BR–BRI1–CNGC12 is essential for high Mn-induced Ca²⁺ influx. (A) A time-course analysis of cytosolic-free calcium concentration [Ca²⁺]_cyt dynamics in 7-d-old WT, cngc12, and bri1-301 seedlings after treatment with 1.5 mM MnCl₂. GFP/mCherry signal was recorded at 15−s intervals. (B) Quantification of the high Mn-induced [Ca²⁺]_cyt changes of A. Data are shown as mean ± SD (n = 3 biologically independent samples). (C) A time-course analysis of cytosolic-free calcium concentration [Ca²⁺]_cyt dynamics in 7-d-old WT seedlings after treatment with 1.5 mM MnCl₂ with or without eBL or BRZ. GFP/mCherry signal was recorded at 15−s intervals. (D) Quantification of the high Mn-induced [Ca²⁺]_cyt changes of C. Data are shown as mean ± SD (n = 3 biologically independent samples).

To investigate BRI1 contributes to high-Mn induced increases in [Ca²⁺]_cyt, we also generated transgenic plants expressing GCaMP6s-mCherry in the bri1-301 background to characterize Mn-triggered calcium signaling dynamics in the transgenic plants. The bri1-301 seedlings also showed a decrease in high-Mn induced calcium signals compared to WT seedlings (Fig. 3 A and B). These results indicate that BRI1 is implicated in high-Mn evoked increases in [Ca²⁺]_cyt in plants. Together, these findings demonstrate that BRI1 positively regulates Mn-triggered calcium signaling, probably via the phosphorylation of CNGC12.

To investigate BR signaling in high-Mn-induced Ca²⁺ flux, GCaMP6s-mCherry/WT plants were treated with epibrassinolide (eBL, an active BR) or brassinazole (BRZ, a BR biosynthesis inhibitor). Monitoring of Mn-elicited cytosolic Ca²⁺ signals revealed that eBL significantly enhanced the high-Mn-induced Ca²⁺ flux, whereas BRZ attenuated it (Fig. 3 C and D). However, neither eBL nor BRZ treatment alone induced detectable Ca²⁺ influx (SI Appendix, Fig. S7 E and F). We further analyzed Ca²⁺ dynamics in BR-related mutants. The bak1-3 serk4-1 double mutant exhibited significantly reduced Mn-triggered Ca²⁺ signals compared to WT (SI Appendix, Fig. S7 G and H). In contrast, the bes1-D and bzr1-1D, as well as the triple mutant bin2-3 bil1 bil2, exhibited no significant differences in Ca²⁺ signaling relative to the WT (SI Appendix, Fig. S7 G–J). This indicates that their Mn-tolerance mechanism likely operates through transcriptional regulation of genes independent of Ca²⁺ signaling pathways.

BRI1 Interacts with CNGC12 In Vivo and In Vitro.

To confirm the interaction between BRI1 and CNGC12, we first conducted a bimolecular fluorescence complementarity (BiFC) experiment to verify the interaction between BRI1 and CNGC12 proteins. The results showed that the cotransformation of BRI1-nYFP and CNGC12-cYFP into tobacco leaves produced fluorescence signals, while the cotransformation of negative control combinations (GUS-nYFP/CNGC12-cYFP, BRI1-nYFP/GUS-cYFP) into tobacco leaves failed to generate fluorescence signals (Fig. 4A), indicating the interaction between BRI1 and CNGC12. To validate their interaction, we performed a firefly luciferase complementation assay (LCI). Cotransformation of BRI1-nLUC and CNGC12-cLUC in tobacco leaves yielded bioluminescence, whereas noninteracting controls (GUS-nLUC/CNGC12-cLUC and BRI1-nLUC/GUS-cLUC) showed no detectable signal (Fig. 4B), demonstrating direct BRI1–CNGC12 interaction. In addition, we conducted reverse transcription PCR (RT-PCR) to confirm that the expression levels of genes (BRI1, CNGC12, and GUS) were consistent in both BiFC and LCI experiments. This validation ensures that the observed interactions are grounded on appropriate expression levels (SI Appendix, Fig. S7 J and K).

Fig. 4.

BRI1 interacts with and phosphorylates CNGC12. (A) BiFC assays demonstrate the interaction between BRI1 and CNGC12. Constructs expressing BRI1-nYFP and CNGC12-cYFP were coexpressed with CBL1-OFP (a PM marker) in N. benthamiana leaves. (Scale bar, 40 μm.) (B) LCI assays of BRI1 with CNGC12. BRI1-nLUC/CNGC12- cLUC were expressed in N. benthamiana leaves for 48 h. GUS-nLUC/CNGC12-cLUC, BRI1-nLUC/GUS-cLUC, and GUS-cLUC/GUS-nLUC were used as negative controls. LCI images were captured using a cooled CCD imaging apparatus. (C) Co-IP assay of BRI1 with CNGC12. CNGC12-Myc/BRI1-FLAG, GUS-Myc/BRI1-FLAG, and CNGC12-Myc/GUS-FLAG, were expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-FLAG agarose beads. Anti-FLAG and anti-Myc antibodies were used to detect the interaction. (D) The amino acid sequence of CNGC12-N. (E) BRI1 phosphorylates CNGC12-N in vitro. Recombinant purified BRI1-KD-His was incubated with GST-CNGC12-N/C in kinase reaction buffer with 1 μCi (γ-³²P) ATP for 30 min at 30 °C. The proteins were separated by SDS-PAGE. Top, autoradiograph; Bottom, CBB staining. The red asterisks denote the kinases BRI1, and the black asterisks indicate the substrates GST and GST-CNGC12-N/C fusion proteins. (F) Ser22 is essential for the phosphorylation of CNGC12 by BRI1. Recombinant purified BRI1-KD-His was incubated with GST-CNGC12-N, or its mutant forms in kinase reaction buffer with 1 μCi (γ-³²P) ATP for 30 min at 30 °C. The proteins were separated by SDS-PAGE. Top, autoradiograph; Bottom, CBB staining. (G) Protein kinase assay of BRI1 with CNGC12-N under high-Mn stress. Ten-day-old seedlings were treated with 1.5 mM MnCl₂ for the indicated time periods. The protein kinases were quantified by western blotting and are shown at the Bottom. (H) Protein kinase assay of BRI1 with CNGC12-N under high-Mn stress. Ten-day-old seedlings were treated with 1.5 mM MnCl₂ with or without eBL or BRZ for the indicated time periods (CK stands for 1.5 mM MnCl₂ treatment alone). The protein kinases were quantified by western blotting and are shown at the Bottom.

To further determine whether BRI1 interacts with CNGC12 in vivo, we performed Coimmunoprecipitation assay (Co-IP) assays. Pro35S:6×Myc-CNGC12/CNGC6 or GUS constructs were transformed into Nicotiana benthamiana leaves in combination with Pro35S:BRI1-3×FLAG, or Pro35S:GUS-3×FLAG constructs. Proteins were immunoprecipitated with anti-FLAG antibody-conjugated agarose and subjected to immunoblot analysis using anti-Myc or anti-FLAG antibodies. CNGC12 coimmunoprecipitated with BRI1 but not with GUS or CNGC6, and GUS did not coimmunoprecipitate with BRI1 either (Fig. 4C). These results demonstrate that BRI1 interacts with CNGC12.

BRI1 Phosphorylates CNGC12 at Ser22 under High-Mn Stress.

The high Mn stress sensitive phenotype of the bri1-301 mutant indicates that the kinase activity of BRI1 is highly associated with plant response to high-Mn stress. Therefore, we explore whether BRI1 regulates Arabidopsis high-Mn-induced Ca²⁺ signaling through phosphorylation modification of CNGC12. We constructed a prokaryotic protein expression vector to fuse the kinase domain of BRI1 with His tag (BRI1-KD-His), as well as the N terminus and C-terminus of CNGC12 with GST tag (GST-CNGC12-N/C). The results in vitro phosphorylation assay results indicated that BRI1 can phosphorylate the N-terminal of CNGC12 in vitro (Fig. 4E).

To investigate potential phosphorylation sites of CNGC12 by BRI1 kinase, we systematically substituted each serine residue in the CNGC12-N domain with alanine through site-directed mutagenesis. Subsequent in vitro kinase assays demonstrated that the S22A substitution (serine-22 to alanine) significantly reduced BRI1-mediated phosphorylation of CNGC12-N (Fig. 4F).

To determine whether phosphorylation of CNGC12 mediated by BRI1 occurs in response to Mn toxicity, we explore the temporal sequence of the phosphorylation event. To this end, C-terminally FLAG-tagged BRI1 overexpression transgenic plants were subjected to 1.5 mM MnCl₂ for 0, 15, 30, 60 min, respectively. The BRI1-FLAG proteins were subsequently extracted and enriched with anti-FLAG antibody-conjugated beads and then incubated with the prokaryotic fusion protein CNGC12-N-GST. We observed that the phosphorylated CNGC12 by BRI1 increased rapidly, peaked at 15 min, and then decreased gradually (Fig. 4G). All together these data show that BRI1 directly phosphorylates CNGC12 at Ser-22 under high-Mn stress in a time-dependent manner.

To investigate the conservation of the Ser residues in positions corresponding to Ser22 in CNGC members, we first performed a phylogenetic analysis of 20 Arabidopsis CNGCs. The comparative analysis reveals that Ser22 is conserved in most of Class I (SI Appendix, Fig. S8A). To investigate whether BRI1-mediated phosphorylation of CNGC12 represents an evolutionarily conserved regulatory mechanism for Mn²⁺ homeostasis, we performed a phylogenetic analysis of CNGC12 homologs across 13 representative species using BLASTP. We then generated a multiple sequence alignment of the N-terminal domains and conducted motif analysis focusing on the conserved serine residue (Ser22) and its flanking regions to assess phosphorylation site conservation (SI Appendix, Fig. S8B). We also performed a phylogenetic analysis of these CNGC12 homologous sequences (SI Appendix, Fig. S8C). These analyses revealed that the S22 BRI1 target motif was conserved in most of the analyzed dicotyledonous species, including Brassica napus, Daucus carota. Remarkably, this Ser22 residue was replaced by an Asp (D) or Glu (E) in monocotyledonous. To validate the conservation of BRI1-mediated phosphorylation at Ser22, we also performed in vitro kinase assays on BRI1-KD and CNGC11-N. These experiments demonstrated that BRI1 also phosphorylates CNGC11 at Ser22 within its N terminus. Notably, substitution of Ser22 with Ala (S22A) significantly reduced phosphorylation levels (SI Appendix, Fig. S7A). Collectively, these findings suggest evolutionary differences in CNGCs regulation between monocots and dicots while underscore the conserved and crucial role of phosphorylation-dependent CNGC12 activation.

BR Enhances the Phosphorylation of CNGC12 by BRI1 under High-Mn Stress.

To establish whether Mn excess activates BRI1 through BR-dependent dimerization with BAK1, we first assessed the kinase activity of BRI1-FLAG under high-Mn stress following eBL or BRZ treatment. BRI1-FLAG transgenic plants were subjected to high-Mn stress while being treated with eBL or BRZ at specified time points. BRI1-FLAG protein was immunoprecipitated using anti-FLAG agarose beads, and phosphorylation assays were performed with CNGC12-N as the substrate. The results demonstrated that eBL treatment significantly enhanced BRI1 kinase activity under high-Mn stress, whereas BRZ treatment attenuated the Mn-induced activation of BRI1 (Fig. 4H).

BAK1 is a key coreceptor in the BR signaling pathway, and the bak1-3 serk4-1 double mutant exhibits a hypersensitive phenotype to high-Mn stress. We also investigated the interaction between BAK1 and CNGC12. However, the results of BiFC and LCI assays indicate that BAK1 does not directly interact with CNGC12 (SI Appendix, Fig. S7 B and C). Furthermore, in vitro phosphorylation assays demonstrate that BAK1 is unable to phosphorylate CNGC12 (SI Appendix, Fig. S7D). These findings suggest that the functional relationship between BAK1 and CNGC12, if any, is likely mediated through indirect mechanisms such as the activity of BRI1 rather than direct physical interaction or phosphorylation.

Although BAK1 does not interact with CNGC12, the bak1-3 serk4-1 double mutant exhibits hypersensitivity to high-Mn stress, suggesting that BAK1 and SERK4 likely modulate Mn tolerance via BRI1-dependent regulation. To test this, we assessed BRI1 kinase activity toward CNGC12 phosphorylation in bak1-3 serk4-1. BRI1-FLAG/bak1-3 serk4-1 material was obtained through crossing. After performing IP to obtain BRI1-FLAG, we conducted phosphorylation assays with CNGC12-N. As previously shown, high-Mn stress enhances BRI1-mediated phosphorylation of CNGC12 in WT plants. In contrast, this stress-induced phosphorylation of CNGC12 by BRI1 kinase was abolished in bak1-3 serk4-1 (SI Appendix, Fig. S7K). Collectively, these data establish that BR signaling and BAK1/SERK4 coreceptors are essential for BRI1-mediated phosphorylation of CNGC12 under Mn stress.

BR-BRI1 Promotes the Ca²⁺ Channel Activity of CNGC12.

Given that BRI1 phosphorylates CNGC12 and CNGC12 functions as a Ca²⁺-permeable channel, we proposed that BRI1-mediated phosphorylation regulates CNGC12-dependent calcium flux. To test this, we performed two-electrode voltage clamp (TEVC) assays in Xenopus oocytes expressing: WT CNGC12, CNGC12 + BRI1, Phospho-dead mutant CNGC12(S22A), CNGC12(S22A) + BRI1 (Fig. 5). In the presence of 30 mM Ca²⁺, we detected distinct inward currents at negative membrane potentials in oocytes expressing CNGC12. These currents were clearly distinguishable from those in control oocytes injected with double distilled water (ddH₂O). When BRI1 was coexpressed with CNGC12 in the oocytes, the inward currents showed a substantial and statistically significant increase compared to those in oocytes expressing CNGC12 alone (Fig. 5 A and B). However, the inward currents in oocytes coexpressing BRI1 with CNGC12^S22A or CNGC12^S22A were comparable to those in oocytes solely expressing CNGC12 (Fig. 5 A and B). These results suggest that BRI1 phosphorylates CNCG12 to promote its Ca²⁺ channel activity.

Fig. 5.

BRI1 promotes the channel activity of CNGC12. (A) Typical whole-cell currents recorded from CNGC12-expressing oocytes in bath solution containing 30 mM Ca²⁺; the water-injected oocytes were used as control. (B) Current–voltage (I–V) curves based on data from A. The data are expressed as means ± SD, with n = 5 for each group. (C) Typical whole-cell currents recorded from CNGC12-expressing oocytes in bath solution containing 30 mM Ca²⁺or 1 nM eBL, the water-injected oocytes were used as control. (D) I–V curves based on data from A. The data are expressed as means ± SD, with n = 5 for each group.

We further assessed epibrassinolide (eBL) modulation of this pathway. TEVC analysis revealed that eBL treatment further enhanced the BRI1-dependent augmentation of CNGC12-mediated Ca²⁺ transport (Fig. 5 C and D), indicating ligand-activated potentiation of the kinase–substrate interaction.

BRI1-Mediated Phosphorylation Is Essential for CNGC12 Function.

To determine how BRI1-mediated phosphorylation regulates CNGC12 function, we analyzed high-Mn stress responses in cngc12 mutants complemented with phospho-null (ProCNGC12:CNGC12^S22A) or phospho-mimetic (ProCNGC 12:CNGC12^S22D) variants expressed at WT-equivalent levels (SI Appendix, Fig. S9). Under high-Mn stress, ProCNGC12:CN GC12^S22A lines exhibited chlorosis, reduced primary root elongation, and decreased chlorophyll content vs. WT and ProCN GC12:CNGC12 controls (Fig. 6 A–D). Conversely, ProCN GC12:CNGC12^S22D lines showed enhanced root elongation and chlorophyll accumulation (Fig. 6 E–H). No phenotypic differences occurred under control conditions (Fig. 6 A–H). Consistent with these phenotypes, Mn quantification revealed higher Mn accumulation in ProCNGC12:CNGC12^S22A plants but lower accumulation in ProCNGC12:CNGC12^S22D lines under Mn stress. These data establish that BRI1-dependent CNGC12 phosphorylation is essential for CNGC12-mediated Mn tolerance (Fig. 6I). These results suggest that the BRI1-mediated CNGC12 phosphorylation is essential for the function of CNGC12 to improve plant Mn toxicity tolerance.

Fig. 6.

BRI1-mediated CNGC12 phosphorylation is vital for its function in Mn toxicity tolerance. (A) Mn toxicity phenotypes of WT, cngc12 mutant, cngc12/ProCNGC12:CNGC12 and cngc12/ProCNGC12:CNGC12^S22A transgenic plants. Four-day-old seedlings were transferred to 1/2 MS medium supplemented with 1.5 mM MnCl₂ for 7 d. (B) Statistical analysis of primary root lengths of plants shown in A (n = 15 seedlings). (C) Statistical analysis of Chla concentration in plants shown in A (n = 5 independent tubes, 3 seedlings/tube). (D) Statistical analysis of Chlb concentration in plants shown in A (n = 5 independent tubes, 3 seedlings/tube). (E) Mn toxicity phenotypes of WT, cngc12 mutant, cngc12/ProCNGC12:CNGC12 and cngc12/ProCNGC12:CNGC12^S22D transgenic plants. The plants were grown and stressed as in A. (F) Statistical analysis of primary root lengths of plants shown in E (n = 15 seedlings). (G) Statistical analysis of Chla concentration in plants shown in E (n = 5 independent tubes, 3 seedlings/tube). (H) Statistical analysis of Chlb concentration in plants shown in E (n = 5 independent tubes, 3 seedlings/tube). (I) Statistical analysis of Mn concentrations in plants shown in A and E (n = 3 biological replicates). (J) A time-course analysis of cytosolic-free calcium concentration [Ca²⁺]_cyt dynamics in 7-d-old WT, cngc12/CNGC12^S22A, and cngc12/CNGC12^S22D seedlings after treatment with 1.5 mM MnCl₂. GFP/mCherry signal was recorded at 15−s intervals. (K) Quantification of the high Mn-induced [Ca²⁺]_cyt changes of J. Data are shown as mean ± SD (Root length, n = 45 seedlings from three biologically independent experiments; Chla/b concentration, n = 15 tubes from three biologically independent experiments; Mn concentrations, 15 samples from three independent experiments). A two-sided t test was used to determine statistical significance, **P < 0.01.

To determine the impact of BRI1-mediated CNGC12 phosphorylation on high-Mn-elicited Ca²⁺ signals, we generated GCaMP6s-mCherry/ProCNGC12:CNGC12^S22A and GCaMP 6s-mCherry/ProCNGC12:CNGC12^S22D lines via genetic crossing. Ca²⁺ imaging revealed that high-Mn-induced Ca²⁺ signals were significantly attenuated in CNGC12^S22A plants compared to WT, whereas signals were enhanced in CNGC12^S22D plants (Fig. 6 J and K). Together, these data demonstrate that BRI1-mediated phosphorylation at Ser22 is critical for CNGC12 function during high-Mn-triggered Ca²⁺ signaling.

BRI1–CNGC12 Regulates High-Mn Response through MTP8 and NRAMP1 Regulation by Ca²⁺ Sensors.

Given that high-Mn-induced Ca²⁺ signals are decoded by the Ca²⁺ sensors CPK5 and CBL1/9-CIPK23, which phosphorylate MTP8 and NRAMP1, respectively, to promote Mn²⁺ sequestration and reduce Mn²⁺ uptake^8,9, we assessed BRI1–CNGC12-dependent regulation of these pathways. Vacuolar Mn²⁺ quantification in cngc12, bri1-301 mutants, and cngc12/CNGC12^S22A lines revealed significant reductions relative to WT, whereas cngc12/CNGC12^S22D showed elevated vacuolar Mn²⁺ accumulation (Fig. 7A). These results establish the BRI1–CNGC12 module as a critical regulator of Mn²⁺ compartmentalization. To determine whether BRI1–CNGC12 signaling affects downstream Ca²⁺ sensor kinase activity, we immunopurified CPK5-FLAG from CPK5-FLAG/bri1-301 and CPK5-FLAG/cngc12 lines. Subsequent in vitro phosphorylation assays using the MTP8 N-terminal domain (MTP8-N) as substrate showed significantly diminished phosphorylation efficiency in both mutants relative to WT controls (Fig. 7B).

Fig. 7.

BRI1–CNGC12 regulate the phosphorylation of Mn²⁺ transporters. (A) Mn and Fe contents in root vacuoles. Vacuoles were isolated from protoplasts of roots exposed to 240 mM MnCl₂ for 1 wk in nutrient solution and analyzed by inductively coupled plasma mass spectrometry (n = 15 samples from three independent experiments), A two-sided t test was used to determine statistical significance, **P < 0.01. (B) Protein kinase assay of CPK5 with MTP8-N under different Mn concentration treatments. Ten-day-old seedlings were treated with 1.5 mM MnCl₂ for the indicated time periods. The protein kinases were quantified by western blotting and are shown at the Bottom. (C) Confocal imaging of roots of transgenic lines expressing a GFP fusion with NRAMP1 in WT or cngc12, bri1-301 mutants. The plants were grown for 5 d in 1/2 MS or with an excess (2 mM) of Mn for 1 h in the presence of 100 μM cycloheximide (CHX). (Scale bar, 6 μm.) (D) Analysis of internal-to-PM signal ratio in (C). Data are presented as mean values ± SD (n = 10). A two-sided t test was used to determine statistical significance, **P < 0.01. (E) The model depicting the function of BRI1 in regulating CNGC12 in Arabidopsis response to high-Mn stress. Mn-activated BRI1 phosphorylates CNGC12 to potentiate its channel activity, triggering a rapid elevation in [Ca²⁺]_cyt. This calcium signature initiates sequential activation of CPKs and CBL–CIPK complexes, which synergistically regulate Mn transporter to enhance the high-Mn stress tolerance.

Furthermore, the high-Mn-induced Ca²⁺ signal activates the CBL1/9-CIPK23 complex, which phosphorylates NRAMP1 to promote its endocytosis^8,36,37. To examine this, we constructed NRAMP1-GFP/bri1-301 and NRAMP1-GFP/cngc12 lines. Confocal microscopy demonstrated that Mn-induced NRAMP1 internalization was markedly impaired in both mutants (Fig. 7 C and D).

Discussion

Unraveling the regulatory role of calcium signaling in plant response to high-Mn stress is a key fundamental question of plant biology. In this study, we revealed that the BRI1–CNGC12 module regulates high-Mn induced Ca²⁺ influx and high-Mn stress tolerance in Arabidopsis. Under high-Mn stress, BRI1 is rapidly activated to phosphorylate CNGC12, which enhances its channel activity, leading to the increase in high-Mn induced Ca²⁺ influx, thus positively regulating the high-Mn stress tolerance ultimately modulating the activity of the Mn transporters MTP8 and NRAMP1 (Fig. 7E).

The CNGC family proteins function as critical calcium channels in plants, with CNGC12 playing pivotal roles in diverse physiological processes in Arabidopsis. Previous studies have demonstrated that the novel chimeric gene AtCNGC11/12 induces multifaceted pathogen resistance responses, including hypersensitive response (HR)-like cell death, while its WT counterparts, AtCNGC11 and AtCNGC12, are also implicated in pathogen defense mechanisms (42). Furthermore, CNGC5/6/9/12 have been shown to be indispensable for ABA-specific Ca²⁺ signaling and ABA-induced stomatal closure in Arabidopsis guard cells (17). In this study, we elucidate the critical role of CNGC12 in manganese-triggered calcium signaling in Arabidopsis, thereby expanding our understanding of its functional mechanisms and highlighting its versatile functionality in plant stress responses and cellular signaling pathways. In addition, similar to the pathogen resistance response process, CNGC11 and CNGC12 also play redundant roles in responding to high-Mn stress.

Accumulating data indicate that protein phosphorylation plays an important role in protein fate during plant responses to biotic and abiotic stresses (43). CNGCs are commonly regulated through phosphorylation. For example, CNGC2 and CNGC4 are activated via phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) (19). During early cold stress, CNGC20 is phosphorylated by PHYTOSULFOKINE RECEPTOR 1 (PSY1R) to enhance its Ca²⁺ transport activity, while at later stages, it undergoes phosphorylation by COLD-RESPONSIVE PROTEIN KINASE 1 (CRPK1), which promotes its degradation (23). OST1 phosphorylates and activates CNGC5/6/9/12, defining the central mechanism through which ABA triggers cytosolic Ca²⁺ signaling in Arabidopsis guard cells (44). Here, we show that CNGC12 activity is likewise activated by BRI1-mediated phosphorylation, highlighting the critical role of phosphorylation in modulating CNGC activity.

Previous studies have established that OST1 phosphorylates Ser-13 of CNGC12 to activate its calcium channel activity (44). Here, we demonstrate that BRI1 enhances the calcium transport activity of CNGC12 through phosphorylation of Ser-22. Both BRI1 and OST1 activate the calcium transport activity of CNGC12 through phosphorylation of its N-terminal region, indicating the critical role of the N terminus in regulating CNGC activity. In addition, the BRI1-mediated phosphorylation of Ser22 in CNGC12 is evolutionarily conserved across both Group I members of the Arabidopsis CNGCs family and CNGC12 orthologs in most of the analyzed dicotyledonous plants, whereas this serine residue is invariably replaced with aspartic acid (D) or glutamic acid (E) in most monocotyledonous species. The activation mechanism mediated by BRI1 phosphorylation of CNGC12 appears evolutionarily conserved in dicotyledonous plants, whereas monocotyledonous species may employ divergent regulatory pathways distinct from those in Arabidopsis and most other dicots.

BRs regulate diverse physiological processes and stress responses in plants (24). Our findings demonstrate that BR signaling mutants exhibit manganese hypersensitivity, while BR application enhances manganese stress tolerance, suggesting an essential role of BR signaling in manganese homeostasis. In addition to its canonical role in growth regulation, BRI1, the BR receptor, has been found to participate in diverse physiological processes (24). BRI1 primarily transduces the BR signal through phosphorylation of BSKs and CDGs (28). In this study, we reveal that BRI1 also phosphorylates the CNGC family calcium channels to modulate high-Mn activated calcium signaling, thereby regulating Mn homeostasis.

The bes1-D, bzr1-1D, and bin2-3 bil1 bil2 mutants exhibit a high-Mn stress tolerance phenotype, indicating that BES1, BZR1, and BIN2/BIL1/BIL2 play critical roles in plant adaptation to Mn excess. However, these mutations did not alter high-Mn-triggered Ca²⁺ signaling, suggesting that their function likely operates through transcriptional regulation of Mn transporters or associated genes to confer tolerance. Future work will identify direct BES1/BZR1 target genes governing high-Mn stress responses. Genetic evidence reveals that CNGC12 mutation abolishes the enhanced Mn toxicity tolerance conferred by BRI1 overexpression. This establishes that BRI1-mediated Ca²⁺ signaling constitutes the primary mechanism underpinning BR-BRI1 induced high-Mn stress tolerance, while BES1/BZR1-driven transcriptional reprogramming serves a secondary role, thus suggesting that functional implementation of BES1/BZR1-mediated transcriptional outputs likely requires concomitant activation of the Ca²⁺ signaling machinery.

Collectively, our findings reveal that the calcium channel CNGC12 and its upstream regulatory kinase BRI1 mediate high-Mn triggered Ca²⁺ signaling in Arabidopsis. The BRI1–CNGC12 module functions as a convergence node bridging BR signaling and Ca²⁺ signaling pathways to coordinate plant adaptation to high-Mn stress in plants.

Materials and Methods

Plant Materials and Growth Conditions.

The Arabidopsis thaliana mutants were described in SI Appendix, Fig. S1 and Table S1. The detailed methods of stress treatment are provided in SI Appendix.

Elemental Analysis.

Seedlings treated with 1.5 mM MnCl₂ in 1/2 MS for 7 d were dried (65 °C) and digested in nitric acid. Elemental analysis used ICP-AES. For vacuolar Mn²⁺, plants were exposed to 240 μM MnSO₄ for 7 d prior to isolation

BiFC Assay.

The BiFC assay was performed as described previously (45). Fluorescence signals were visualized by confocal microscopy.

Split-LCI Assay.

The LCI assay was performed as described previously (45). The signals were detected by a charge-coupled device (CCD) (Princeton, Lumazone Pylon 2048B).

Co-IP Assays.

The Co-IP assays were performed, as described previously (7). The interactions were analyzed by immunoblotting using anti-Myc/anti-FLAG antibodies.

Protein Kinase Assays.

In vitro kinase assays were performed by incubating CNGC12-N/C (WT/mutant) with BRI1-KD-His in reaction buffer. Phosphorylated proteins detected by SDS-PAGE/autoradiography.

Electrophysiological Assays.

Voltage-clamp recordings in Xenopus oocytes followed published methods (23). cRNAs transcribed from pNB1 vectors were injected. Currents were recorded with hyperpolarized pulses.

Ratiometric Ca²⁺ Imaging.

Ratiometric Ca²⁺ imaging used GCaMP6s-mCherry-expressing plants grown in Hoagland medium for 5 d. Imaging in Hoagland buffer with 1.5 mM MnSO₄ (HMn) was performed on a confocal microscope (45).

Chlorophyll Content Measurement.

Fresh leaves were immersed in 95% (v/v) ethanol and incubated in darkness for 16 h at room temperature. Absorbance of the extracts was measured at 665 nm (A₆₆₅) and 649 nm (A₆₄₉) using a spectrophotometer.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

Study data are included in the article and/or SI Appendix.