Abstract
Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2–BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL–CIPK) and Ca2+-independent (FLS2–BAK1–BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.
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Data availability
All the data generated in this study are available in the paper and its Supplementary Information (Supplementary Tables 1–3). Uncropped gel and blot images are provided in Supplementary Fig. 1. The crystal structure of Ca2+-associated ScVCX1 was retrieved from PDB:4K1C. Source data are provided with this paper.
Code availability
No customized code was generated in this study.
References
White, P. J. & Broadley, M. R. Calcium in plants. Ann. Bot. 92, 487–511 (2003).
Clapham, D. E. Calcium signaling. Cell 131, 1047–1058 (2007).
Luan, S. & Wang, C. Calcium signaling mechanisms across kingdoms. Annu. Rev. Cell Dev. Biol. 37, 311–340 (2021).
Wang, C. & Luan, S. Calcium homeostasis and signaling in plant immunity. Curr. Opin. Plant Biol. 77, 102485 (2023).
Jones, J. D. G. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006).
Tian, W. et al. A calmodulin-gated calcium channel links pathogen patterns to plant immunity. Nature 572, 131–135 (2019).
Thor, K. et al. The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity. Nature 585, 569–573 (2020).
Bi, G. et al. The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling. Cell 184, 3528–3541 (2021).
Jacob, P. et al. Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels. Science 373, 420–425 (2021).
Bjornson, M., Pimprikar, P., Nürnberger, T. & Zipfel, C. The transcriptional landscape of Arabidopsis thaliana pattern-triggered immunity. Nat. Plants 7, 579–586 (2021).
Ast, C. et al. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins. Nat. Commun. 8, 431 (2017).
Bose, J., Pottosin, I. I., Shabala, S. S., Palmgren, M. G. & Shabala, S. Calcium efflux systems in stress signaling and adaptation in plants. Front. Plant Sci. 2, 85 (2011).
Cheng, N.-H. et al. Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis. Plant Physiol. 138, 2048–2060 (2005).
Boursiac, Y. et al. Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway. Plant Physiol. 154, 1158–1171 (2010).
Hilleary, R. et al. Tonoplast-localized Ca2+ pumps regulate Ca2+ signals during pattern-triggered immunity in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 117, 18849–18857 (2020).
Rahmati Ishka, M. et al. Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness. Plant Physiol. 185, 1966–1985 (2021).
Li, Z., Harper, J. F., Weigand, C. & Hua, J. Resting cytosol Ca2+ level maintained by Ca2+ pumps affects environmental responses in Arabidopsis. Plant Physiol. 191, 2534–2550 (2023).
Conn, S. J. et al. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis. Plant Cell 23, 240–257 (2011).
Pittman, J. K. & Hirschi, K. D. Regulation of CAX1, an Arabidopsis Ca2+/H+ antiporter. Identification of an N-terminal autoinhibitory domain. Plant Physiol. 127, 1020–1029 (2001).
Waight, A. B. et al. Structural basis for alternating access of a eukaryotic calcium/proton exchanger. Nature 499, 107–110 (2013).
Xu, S.-L. et al. Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis. Proc. Natl Acad. Sci. USA 114, E1536–E1543 (2017).
Obayashi, T., Hibara, H., Kagaya, Y., Aoki, Y. & Kinoshita, K. ATTED-II v11: a plant gene coexpression database using a sample balancing technique by subagging of principal components. Plant Cell Physiol. 63, 869–881 (2022).
Tang, R. J. et al. Tonoplast CBL–CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis. Proc. Natl. Acad. Sci. USA 112, 3134–3139 (2015).
Tang, R.-J., Wang, C., Li, K. & Luan, S. The CBL–CIPK calcium signaling network: unified paradigm from 20 years of discoveries. Trends Plant Sci. 25, 604–617 (2020).
Liu, J., Ishitani, M., Halfter, U., Kim, C. S. & Zhu, J. K. The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc. Natl Acad. Sci. USA 97, 3730–3734 (2000).
Koster, P., DeFalco, T. A. & Zipfel, C. Ca2+ signals in plant immunity. EMBO J. 41, e110741 (2022).
Yu, X. et al. A phospho-switch constrains BTL2-mediated phytocytokine signaling in plant immunity. Cell 186, 2329–2344 (2023).
Zhao, C. et al. A mis-regulated cyclic nucleotide-gated channel mediates cytosolic calcium elevation and activates immunity in Arabidopsis. New Phytol. 230, 1078–1094 (2021).
Grant, M. et al. The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death. Plant J. 23, 441–450 (2000).
Ranf, S., Eschen-Lippold, L., Pecher, P., Lee, J. & Scheel, D. Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns. Plant J. 68, 100–113 (2011).
Ranf, S. et al. Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1. BMC Plant Biol. 14, 374 (2014).
Liu, F. et al. The activated plant NRC4 immune receptor forms a hexameric resistosome. Preprint at bioRxiv https://doi.org/10.1101/2023.12.18.571367 (2023).
Ngou, B. P. M., Ahn, H.-K., Ding, P. & Jones, J. D. G. Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature 592, 110–115 (2021).
Yuan, M. et al. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature 592, 105–109 (2021).
Li, L. et al. The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe 15, 329–338 (2014).
Lu, D. et al. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc. Natl Acad. Sci. USA 107, 496–501 (2010).
Ma, X. et al. Ligand-induced monoubiquitination of BIK1 regulates plant immunity. Nature 581, 199–203 (2020).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).
Wang, Y. et al. CNGC2 is a Ca2+ influx channel that prevents accumulation of apoplastic Ca2+ in the leaf. Plant Physiol. 173, 1342–1354 (2017).
Zhang, Y. & Li, X. Salicylic acid: biosynthesis, perception, and contributions to plant immunity. Curr. Opin. Plant Biol. 50, 29–36 (2019).
Ngou, B. P. M., Jones, J. D. G. & Ding, P. Plant immune networks. Trends Plant Sci. 27, 255–273 (2022).
Fu, Z. Q. & Dong, X. Systemic acquired resistance: turning local infection into global defense. Annu. Rev. Plant Biol. 64, 839–863 (2013).
Wildermuth, M. C., Dewdney, J., Wu, G. & Ausubel, F. M. Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414, 562–565 (2001).
Fu, Z. Q. et al. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486, 228–232 (2012).
Ding, Y. et al. Opposite roles of salicylic acid receptors NPR1 and NPR3/NPR4 in transcriptional regulation of plant immunity. Cell 173, 1454–1467 (2018).
Cui, H. et al. A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity. New Phytol. 213, 1802–1817 (2017).
He, Z., Webster, S. & He, S. Y. Growth–defense trade-offs in plants. Curr. Biol. 32, R634–R639 (2022).
Catalá, R. et al. Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis. Plant Cell 15, 2940–2951 (2003).
Zhao, J., Barkla, B. J., Marshall, J., Pittman, J. K. & Hirschi, K. D. The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity. Planta 227, 659–669 (2008).
Yang, J. et al. The vacuolar H+/Ca transporter CAX1 participates in submergence and anoxia stress responses. Plant Physiol. 190, 2617–2636 (2022).
Hwang, I., Harper, J. F., Liang, F. & Sze, H. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain. Plant Physiol. 122, 157–168 (2000).
Yang, D.-L. et al. Calcium pumps and interacting BON1 protein modulate calcium signature, stomatal closure, and plant immunity. Plant Physiol. 175, 424–437 (2017).
Xiang, T. et al. Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Curr. Biol. 18, 74–80 (2008).
Yu, H., Yan, J., Du, X. & Hua, J. Overlapping and differential roles of plasma membrane calcium ATPases in Arabidopsis growth and environmental responses. J. Exp. Bot. 69, 2693–2703 (2018).
Capieaux, E., Vignais, M. L., Sentenac, A. & Goffeau, A. The yeast H+-ATPase gene is controlled by the promoter binding factor TUF. J. Biol. Chem. 264, 7437–7446 (1989).
Mumberg, D., Müller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995).
Cunningham, K. W. & Fink, G. R. Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2226–2237 (1996).
Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).
Acknowledgements
We thank K. D. Hirschi for providing the cax1-1 cax3-1 double mutant; W. B. Frommer and T. Kleist for the MatryoshCaMP6s calcium reporter line; J.-M. Zhou for the fls2, bak1-4 and bik1 pbl1 mutants; J. E. Parker for the eds1-2 (Col) mutant; J. F. Harper for the aca4-3 aca11-5 and aca1-7/2-3/7-5 mutants; J. Hua for the aca8 aca10 mutant; Y. Zhang for the npr1-1 npr4-4D mutant seeds; K. D. Hirschi and C. Chang for the yeast strain K667; B. Staskawicz for the P. syringae pv. tomato DC3000, Pst DC3000 (avrRpt2) and Pst DC3000 (avrRps4) strains; M. C. Wildermuth for the hrcC− strain; L. Shan and P. He for the BIK1-9KR plasmid; Z. Wang and S. Xu for sharing the Arabidopsis phospho-proteomics data; and E. Calvanese for lab assistance. This work was supported by the National Institutes of Health (R01GM138401 to S.L.), the National Science Foundation (MCB-2041585 to S.L.) and the California Agriculture Experimental Station. We thank P. He and M. West of the High-Throughput Screening Facility at UC Berkeley for assistance in the use of the Perkin-Elmer Envision Multilabel Plate Reader. The Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at the University of California, Berkeley, was supported in part by NIH S10 Instrumentation Grant S10RR025622. C.W. acknowledges support from the Tang Distinguished Scholarship managed by the University of California, Berkeley.
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The work was conceptualized by C.W., R.-J.T. and S.L.; the methodology was designed by C.W. and R.-J.T.; the investigation was done by C.W., R.-J.T., S.K., Y.L., K.R. and A.V.; the structure was modelled by X.X.; the formal analysis was by C.W., R.-J.T. and S.L.; the visualization was by C.W. and S.L.; the original draft was written by C.W., R.-J.T. and S.L.; it was read and edited by S.L., C.W. and R.-J.T.; funding acquisition was done by S.L.; and supervision was by S.L.
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Extended data figures and tables
Extended Data Fig. 1 Phenotypic analysis of aca and cax mutants.
a, PCR-based genotyping of cax mutants. LP/RP, primers matching genomic sequences at left and right borders of T-DNA insertions. LB, primer matching the left border of T-DNA sequence. b, Growth phenotypes of cax mutants under indicated conditions. c, RT-PCR analysis of full-length transcripts of CAX1 and CAX3. The ACTIN was used as an internal reference. Data in a, c are representative results from two independent experiments. d, Schematic presentation of T-DNA insertions in cax1 and cax3 mutants. e-g, Growth phenotypes of various aca mutants under indicated conditions. h, j, [Ca2+]cyt dynamics (R) upon the addition of 10 mM CaCl2 (arrow). Curves were plotted with mean values. i, k, Quantitative analysis of the data as in h, j. R(0Ca) and R (10Ca) indicate [Ca2+]cyt before and at 60 min after Ca2+ treatment. Bar graphs show individual data points scattered around mean values. Asterisks (***) indicate significant differences (P < 0.0001) resulted from One-way ANOVA multiple comparison tests between wild-type and mutants. ns, not significant. n = 18, 18, 18, 10, 18, 18, 10, 10, 10, 16, 17, 18, 18 (h, i) and n = 19, 16, 29, 35, 16, 16, 24 (j, k) leaf discs from three independent experiments. l, Growth phenotypes of 3-week-old plants grown on agar media containing indicated concentrations of CaCl2. m, Total calcium content in the leaves of soil-grown plants. n = 4 extracts. DW, dry weight. n, Normalized [Ca2+]cyt dynamics in cax1 cax3 mutant upon the addition of 5 mM CaCl2 (arrow). Data are mean ± s.e.m. n = 10 leaf discs in each of two independent experiments. o, Growth phenotypes of 18-day-old cax1 cax3 mutant plants grown on agar media containing indicated concentrations (mM) of CaCl2 and KCl. p, Bar graph showing rosette diameters as means ± s.e.m. n = 24 plants from 3 plates as in (o). P values resulted from One-way ANOVA multiple comparison tests between wild type and mutants. ns, not significant. Scale bars = 5 cm for b, e, f, g, i, o. Photos show representative plants from at least two independent experiments (a-g, l, o).
Extended Data Fig. 2 Sequence and expression analysis of Arabidopsis CAX1/3 transporters.
a, b, Predicted topological structures of the Arabidopsis CAX1/3 and a yeast Ca2+/H+ exchanger (S. cerevisiae VCX1). The N-terminal auto-inhibitory domain highlighted in plant CAXs (a) is absent in yeast VCX1 (b). c, Sequence alignment of auto-inhibitory domain of CAX1/3 orthologs from plant species including dicots, monocots and a basal angiosperm. A conserved serine (S)-cluster is highlighted and asterisked. d, In planta phosphorylation sites of CAX1/3. e, A list of genes co-expressing with CAX1. Kinases with particular interests are highlighted. f, Quantitative real-time PCR analysis of transcript levels of CBL2, CBL3, CIPK3, CIPK9, CIPK26, CAX1 and CAX3 in various plant tissues. Data are mean ± s.d. n = 3 plant samples. The expression levels were normalized to a reference gene ACTIN.
Extended Data Fig. 3 CBL-CIPK-mediated CAX1/3 activation requires functional CBL-CIPK modules and is conserved in land plants.
a, A phylogenetic tree of the CBL family. PM, plasma membrane. b, Constructs for co-expressing multiple proteins in yeast. Pro, Promoter, Ter, Terminator, CDS, coding sequences. Asterisks, stop codons. c, d, Yeast functional assay on high Ca2+ medium using the Ca2+-sequestration deficient yeast strain K667 expressing different CAX variants with or without CBL-CIPK modules. CAX1/3 Δ63, truncated CAX1/3 without the N-terminal auto-inhibitory domain (63 amino acids). Ev, empty vector. e, Schematic diagrams of CIPK9 variants. CIPK9-K48N and CIPK9-T178D, kinase-dead and constitutively active forms of CIPK9. CIPK9ΔNAF, truncated CIPK9 without CBL-interacting domain. f-i, Yeast functional assay using the yeast strain K667 co-expressing CBL3-CIPK9 variants with CAX1/3 (f, g), or CBL-CIPK modules and CAXs from different species (h, i). Yeast functional assays (a-i) are repeated two times. j, Growth phenotypes of soil-grown plants. k, Growth phenotypes of plants under high-Na+ and high-Ca2+ conditions. l, Growth phenotypes of wild-type and cipk mutants in hydroponic solutions containing 0.1 mM or 5 mM CaCl2. Photographs were taken 2 weeks (k) and 4 weeks (l) after 7-day-old seedlings were transferred to agar media (k) or hydroponic cultures (l). Representative photos from three independent experiments are shown. Scale bars = 2 cm for j-l. m, [Ca2+]cyt kinetics following the addition of 10 mM CaCl2 (arrow). n, Statistical analysis of [Ca2+]cyt before or after Ca2+ treatment as in m. Data in m, n are means ± s.e.m. n = 12 (cax1-1 cax3-1) and n = 24 (Col-0, cbl2 cbl3, cipk3/9/26) leaf discs from two independent experiments. P values resulted from One-way ANOVA multiple comparison tests between wild type and mutants.
Extended Data Fig. 4 Phosphorylation of the S-cluster in CAX1/3 is required for normal plant growth in the soil.
a, b, In vitro phosphorylation of CAX1/3 by CIPK3/9/26 kinases. CIPK9-K48N, kinase-dead mutant; CIPK9-T178D constitutively active form. N63, N-terminal region of 63 amino acids in CAX1/3. CBB, Coomassie Brilliant Blue stained gels. Autorad., autoradiograph. Numbers on the right side of the gels indicate molecular weights (KD). Images are representatives of three independent experiments. c, d, Identification of phosphorylated amino acid residues of CAX1/3 by mass spectrometry. The phosphorylated residues are asterisked and the conserved S-cluster is highlighted. e, f, Phenotypes of soil-grown plants. The cax1-1 cax3-1 mutant plants were transformed with either wild-type (WT-L1, 2) or S-cluster mutated (4A-L1, 2) CAX1 driven by the CAX1 native promoter. L1, 2 represent two independent transgenic lines. Rosette diameters of multiple plants in e are shown in f. Bar graph shows individual data points scattered around means ± s.d. Letters (a, b, c) indicate statistical differences (P < 0.005, n = 6 plants), resulted from One-way ANOVA comparison tests among different genotypes. Experiments are repeated three times. Scale bar = 2 cm in e.
Extended Data Fig. 5 Ca2+-dependent regulation of CAX1/3 by the tonoplast CBL-CIPK complex.
a, Schematic depiction of four EF-hand motifs in three Ca2+ sensors. Asterisks denote the critical Glu (E) residues required for Ca2+ binding. M1-M4 denotes E to Gln (Q) mutation. b, Mobility shift of different Ca2+ sensors in response to Ca2+ in a native PAGE gel. The image is a representative of five independent experiments. c, In vitro phosphorylation of CAX1/3 by CBL3-CIPK9 modules in a Ca2+-dependent manner. CBB, Coomassie Brilliant Blue stained gel. Autorad., autoradiograph. Numbers on the right side of the gels indicate molecular weights (KD). Images are representatives of three independent experiments. d, Yeast functional assay using yeast strain K667 co-expressing CBL3 variants with CIPK9 and CAX1/3. CBL3-qM, CBL3 variant with E to Q mutations in all four EF-hand motifs. Images are representatives of two independent experiments. e, Growth phenotypes of wild type, cbl2 cbl3, and complemented plants in response to external Ca2+ in hydroponics. Photos were taken five weeks after seedlings were transferred to the hydroponic cultures. Representative pictures show two independent transgenic lines expressing CBL3 (WT-L1 and WT-L2) or CBL3-qM (qM-L1 and qM-L2) in the cbl2 cbl3 background. f, Statistical analysis of shoot biomass as in e. Bar graphs show individual data points scattered around the mean values. Letters (a, b) indicate statistically significant differences (p < 0.0001 from One-way ANOVA comparison tests among different genotypes in each group, n = 6 plants.
Extended Data Fig. 6 External Ca2+ and bacterial invasions elicit cytosolic Ca2+ changes.
a-c, Flg22-triggered [Ca2+]cyt elevation in plants of various genotypes. Data are mean values (a) and means ± s.e.m. (b, c). n = 24 leaf discs from three independent experiments. d, e, [Ca2+]cyt kinetics upon cold treatment by adding ice water (arrow). n = 12 leaf discs from two independent experiments. Data are means ± s.e.m. P values (b, c, e) resulted from One-way ANOVA between wild type and mutants. ns, not significant. f, g, [Ca2+]cyt dynamics after leaf infiltrations with bacterial strains. f, [Ca2+]cyt kinetics over a 7 h period. g, Quantitative analysis of data at 4 h as in f. Data in g are means ± s.e.m. n = 10 leaf discs from two independent experiments. P values resulted from One-way ANOVA between bacteria- and water-infiltrated samples. h, [Ca2+]cyt dynamics after leaf infiltration with either water or bacterial strain Pst hrcC−. i, Normalized [Ca2+]cyt dynamics of bacterium-infiltrated versus water-infiltrated leaf discs as in h. Data are means ± s.e.m. n = 12 leaf discs from two independent experiments. j, k, Time course and statistical analysis of [Ca2+]cyt dynamics following the addition of different concentrations of external CaCl2 (mM) (arrows). Data are means ± s.d., n = 24 leaf discs from three independent experiments. P values resulted from One-way ANOVA comparing cncg2 mutant with wild type. l-o, [Ca2+]cyt changes of water or bacterium-infiltrated leaf discs in the presence or absence of 10 mM CaCl2 solution. l, n, [Ca2+]cyt kinetics over a 7 h period. Data are means. m, o, Statistical analysis of data at indicated time point as in l, n. Data are means ± s.e.m. n = 6 leaf discs in each of two independent experiments. P values resulted from One-way ANOVA between indicated groups. Bar graphs presented in this figure show individual data points scattered around mean values.
Extended Data Fig. 7 BIK1 and PBL1 phosphorylate CAX1/3 at the S-cluster in the auto-inhibitory domain.
a-c, Flg22-triggered CAX1/3 phosphorylation in Arabidopsis mesophyll protoplasts. d, Flg22-triggered phosphorylation in CAX1/3 but not CAX2/4/5/6. e, Flg22-triggered CAX1 phosphorylation in wild-type and cbl2 cbl3 protoplasts. f-i, Flg22-induced phosphorylation of CAX1/3 in protoplasts overexpressing the wild type or kinase-dead form of BIK1 (f, g) or PBL1 (h, i). j, In vitro phosphorylation of CAX1/3 by PBL1 or BIK1. CBB, Coomassie Brilliant Blue stained gel. Autorad., autoradiograph. k, Identification of BIK1-mediated phosphorylation sites in CAX1/3 using mass spectrometry. l, m, S-cluster in CAX3 is essential for flg22-triggered, BIK1-dependent phosphorylation in bik1 pbl1 mutant background. Quantitative values are normalized phosphorylation ratios. n = 3 biological repeats. In a-j, l, m, CAX1/3 N indicates CAX1/3 N-terminal region containing two transmembrane domains. Phosphorylated (p) and non-phosphorylated (np) forms of CAXs were indicated on Phos-tag (Phos.), or on regular (Reg.) SDS-PAGE gels. RbcL., Rubisco large subunit. Numbers on the right side of the gels indicate protein molecular weight (KD). Presented in this figure are representative data from two (d, h, i) or three (a-c, e-g, j, l, m) independent experiments.
Extended Data Fig. 8 Flg22-triggered and BIK1- or PBL1-dependent Ca2+ efflux.
a-d, Flg22-triggered Ca2+ efflux in a dose-dependent manner. The [Ca2+]cyt dynamics (R/R0) were normalized against the values prior to addition of flg22 (arrows). D1 and D2 indicate two distinct drops of [Ca2+]cyt, reflecting Ca2+ efflux. Blue lines in a-d indicate [Ca2+]cyt dynamics induced by 100 mM CaCl2 solution without flg22. Data (a-d) are mean values. n = 8 leaf discs in each of the two independent experiments. e, Statistical analysis of data points for D1 and D2 as shown in b-d. P values resulted from One-way ANOVA comparing indicated groups. f-i, Ca2+ efflux in various mutants triggered by 100 nM or 1000 nM flg22 (arrows). n = 16 (fls2) and n = 24 (Col-0, bak1-4 and bik1 pbl1) leaf discs from two independent experiments. j, Statistical analysis of D1 and D2 as shown in f-i. P values resulted from One-way ANOVA multiple comparison tests between wild type and mutants. k, BIK1 and PBL1 act redundantly in flg22-induced Ca2+ efflux. Data are means ± s.e.m. n = 12 leaf discs in each of two independent experiments. l, Statistical analysis of D2 as shown in k. Bar graphs presented in this figure show individual data points scattered around mean values. P values resulted from One-way ANOVA multiple comparison tests between wild-type and mutant plants.
Extended Data Fig. 9 BIK1 and PBL1 regulate Ca2+ efflux during PTI, but not in response to external Ca2+ elevation.
a, b, Time courses and statistical analysis of [Ca2+]cyt changes after leaf infiltrations with either water or Pst hrcC−. Data are means ± s.e.m (a) and Box & Whiskers graphs (b). Whiskers cover minimum to maximum values, and boxes extend from 25th to 75th percentiles with median values indicated. n = 18 leaf discs from three independent experiments. P values and significant differences (indicated by a, b, a’, b’) resulted from Two-way ANOVA multiple comparison tests between water- and bacterium-infiltrated samples. c, Growth phenotypes of soil-grown plants after spray inoculation with Pst hrcC− at OD600 = 0.2. Two-week-old plants were inoculated, and pictures were taken 12 days after inoculation. Scale bars = 5 cm. d, Shoot fresh weight of the plants shown in c. Data are mean ± s.e.m. n = 16 plants in each of two independent experiments. P values resulted from Two-way ANOVA multiple comparison tests between bacterium-treated and control samples. The bik1 pbl1 mutant was more inhibited than the wild type by the bacteria. e, Growth phenotypes in response to increasing external Ca2+. f, Statistical analysis of rosette diameters. n = 16 plants from three plates. P values resulted from Two-way ANOVA multiple comparison tests between wild type and mutants within each group. Scale bar = 2 cm. g-j, Time courses of [Ca2+]cyt (g) and normalized [Ca2+]cyt dynamics (i) following the addition of 10 mM CaCl2 (arrow), and statistical analysis (h,j) for data points as in g, i. Data are means ± s.e.m. n = 9 (cax1-3 cax3-3) and n = 11 (Col-0 and bik1 pbl1) leaf discs in each of three independent experiments. P values resulted from One-way ANOVA multiple comparison tests between wild type and mutants.
Extended Data Fig. 10 Cytosolic Ca2+ elevation inhibits growth largely through SA signaling pathway.
a, Transcript levels of defense-related genes in wild-type and cax1 cax3 mutant plants grown in soil for three weeks or in hydroponic conditions. For the hydroponic growth, plants were first grown in low Ca2+ (0.1 mM) medium for three weeks, followed by a medium replacement with either a low Ca2+ (0.1 mM) or high Ca2+ (10 mM) medium for another two days. Heatmap displays the relative expression level of each gene in cax1 cax3 mutants as compared to wild-type plants. b, Transcript levels of defense-related genes in leaves infiltrated with Pst hrcC− and Pst DC3000 (avrRps4) at OD600 = 0.2, inducing PTI and ETI, respectively. Heatmap displays the relative expression level of each gene in bacterium-infiltrated samples compared to the water-infiltrated sample. c, Growth phenotypes of 3-week-old plants grown in autoclaved soil. Scale bar = 2 cm. d, Statistical analysis of rosette diameters as in c. Data are mean ± s.e.m. n = 8 plants in each of three independent experiments. e, Time courses of normalized [Ca2+]cyt dynamics (R/R0, in which R0 indicates data prior treatments) following addition of 10 mM CaCl2 solution (arrows). Data are means ± s.e.m. n = 24 leaf discs from three independent experiments. f, Statistical analysis of the data at 30 min as in e. Different letters (a, b, a’, b’) denote statistically significant differences within each group (P < 0.01 for d, P < 0.0001 for f). For d, f, P values are resulted from One-way ANOVA multiple comparison tests among different genotypes within each group. Bar graphs (d, f) show individual data points scattered around means.
Supplementary information
Supplementary Fig. 1
Uncropped gel and blot images.
Supplementary Table 1
A full list of co-expression genes with CAX1.
Supplementary Table 2
A full list of plant materials and reporter lines.
Supplementary Table 3
Plasmids and oligonucleotides.
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Wang, C., Tang, RJ., Kou, S. et al. Mechanisms of calcium homeostasis orchestrate plant growth and immunity. Nature 627, 382–388 (2024). https://doi.org/10.1038/s41586-024-07100-0
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DOI: https://doi.org/10.1038/s41586-024-07100-0
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