A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis

Article metrics


Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca2+-mediated stress signals remains poorly understood1,2. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca2+ flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca2+ dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca2+ in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: cMCU localizes to chloroplasts in Arabidopsis mesophyll cells.
Fig. 2: Recombinant cMCU mediates Ca2+ fluxes in electrophysiological experiments and in a heterologous expression system.
Fig. 3: Monitoring of stromal Ca2+ concentration reveals differential calcium dynamics and signalling in wild-type versus cMCU knockout plants.
Fig. 4: Plants lacking cMCU are drought resistant and recover quickly following rewatering.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.


  1. 1.

    Kudla, J. et al. Advances and current challenges in calcium signaling. New Phytol. 218, 414–431 (2018).

  2. 2.

    Zhu, J. K. Abiotic stress signaling and responses in plants. Cell 167, 313–324 (2016).

  3. 3.

    Leister, D., Wang, L. & Kleine, T. Organellar gene expression and acclimation of plants to environmental stress. Front. Plant Sci. 8, 387 (2017).

  4. 4.

    Dodd, A. N., Kudla, J. & Sanders, D. The language of calcium signaling. Annu. Rev. Plant Biol. 61, 593–620 (2010).

  5. 5.

    Kmiecik, P., Leonardelli, M. & Teige, M. Novel connections in plant organellar signalling link different stress responses and signalling pathways. J. Exp. Bot. 67, 3793–3807 (2016).

  6. 6.

    Nomura, H. & Shiina, T. Calcium signaling in plant endosymbiotic organelles: mechanism and role in physiology. Mol. Plant 7, 1094–1104 (2014).

  7. 7.

    Guo, H. et al. Plastid–nucleus communication involves calcium-modulated MAPK signalling. Nat. Commun. 7, 12173 (2016).

  8. 8.

    Sai, J. & Johnson, C. H. Dark-stimulated calcium ion fluxes in the chloroplast stroma and cytosol. Plant Cell 14, 1279–1291 (2002).

  9. 9.

    Stael, S. et al. Plant organellar calcium signalling: an emerging field. J. Exp. Bot. 63, 1525–1542 (2012).

  10. 10.

    Hochmal, A. K., Schulze, S., Trompelt, K. & Hippler, M. Calcium-dependent regulation of photosynthesis. Biochim. Biophys. Acta 1847, 993–1003 (2015).

  11. 11.

    Loro, G. et al. Chloroplast-specific in vivo Ca2+ imaging using yellow cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma. Plant Physiol. 171, 2317–2330 (2016).

  12. 12.

    Sello, S. et al. Dissecting stimulus-specific Ca2+ signals in amyloplasts and chloroplasts of Arabidopsis thaliana cell suspension cultures. J. Exp. Bot. 67, 3965–3974 (2016).

  13. 13.

    Nomura, H. et al. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat. Commun. 3, 926 (2012).

  14. 14.

    Costa, A., Navazio, L. & Szabo, I. The contribution of organelles to plant intracellular calcium signalling. J. Exp. Bot. 69, 4175–4193 (2018).

  15. 15.

    Carraretto, L. et al. Ion channels in plant bioenergetic organelles chloroplast and mitochondria: from molecular identification to function. Mol. Plant 9, 371–395 (2016).

  16. 16.

    Carraretto, L. et al. A thylakoid-located two-pore K+ channel controls photosynthetic light utilization in plants. Science 342, 114–118 (2013).

  17. 17.

    De Stefani, D., Raffaello, A., Teardo, E., Szabo, I. & Rizzuto, R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476, 336–340 (2011).

  18. 18.

    Teardo, E. et al. Physiological characterization of a plant mitochondrial calcium uniporter in vitro and in vivo. Plant Physiol. 173, 1355–1370 (2017).

  19. 19.

    Kreimer, G., Melkonian, M., Holtum, J. A. & Latzko, E. Characterization of calcium fluxes across the envelope of intact spinach chloroplasts. Planta 166, 515–523 (1985).

  20. 20.

    Nguyen, N. X. et al. Cryo-EM structure of a fungal mitochondrial calcium uniporter. Nature 559, 570–574 (2018).

  21. 21.

    Yoo, J. et al. Cryo-EM structure of a mitochondrial calcium uniporter. Science 361, 506–511 (2018).

  22. 22.

    Baradaran, R., Wang, C., Siliciano, A. F. & Long, S. B. Cryo-EM structures of fungal and metazoan mitochondrial calcium uniporters. Nature 559, 580–584 (2018).

  23. 23.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

  24. 24.

    McAinsh, M. R. & Pittman, J. K. Shaping the calcium signature. New Phytol. 181, 275–294 (2009).

  25. 25.

    Mehlmer, N. et al. A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana. J. Exp. Bot. 63, 1751–1761 (2012).

  26. 26.

    Sello, S. et al. Chloroplast Ca2+ fluxes into and across thylakoids revealed by thylakoid-targeted aequorin probes. Plant Physiol. 177, 38–51 (2018).

  27. 27.

    Kazama, D., Kurusu, T., Mitsuda, N., Ohme-Takagi, M. & Tada, Y. Involvement of elevated proline accumulation in enhanced osmotic stress tolerance in Arabidopsis conferred by chimeric repressor gene silencing technology. Plant Signal. Behav. 9, e28211 (2014).

  28. 28.

    Shkolnik, D., Nuriel, R., Bonza, M. C., Costa, A. & Fromm, H. MIZ1 regulates ECA1 to generate a slow, long-distance phloem-transmitted Ca2+ signal essential for root water tracking in Arabidopsis. Proc. Natl Acad. Sci. USA 115, 8031–8036 (2018).

  29. 29.

    Choudhury, F. K., Rivero, R. M., Blumwald, E. & Mittler, R. Reactive oxygen species, abiotic stress and stress combination. Plant J. 90, 856–867 (2017).

  30. 30.

    de Zelicourt, A., Colcombet, J. & Hirt, H. The role of MAPK modules and ABA during abiotic stress signaling. Trends Plant Sci. 21, 677–685 (2016).

  31. 31.

    Ichimura, K., Mizoguchi, T., Yoshida, R., Yuasa, T. & Shinozaki, K. Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J. 24, 655–665 (2000).

  32. 32.

    Lee, K. et al. Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis. J. Biol. Chem. 283, 23581–23588 (2008).

  33. 33.

    Brock, A. K. et al. The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression. Plant Physiol. 153, 1098–1111 (2010).

  34. 34.

    Meng, X. & Zhang, S. MAPK cascades in plant disease resistance signaling. Annu Rev. Phytopathol. 51, 245–266 (2013).

  35. 35.

    Vogel, M. O. et al. Fast retrograde signaling in response to high light involves metabolite export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF transcription factors in Arabidopsis. Plant Cell 26, 1151–1165 (2014).

  36. 36.

    Dubois, M. et al. The ETHYLENE RESPONSE FACTORs ERF6 and ERF11 antagonistically regulate mannitol-induced growth inhibition in Arabidopsis. Plant Physiol. 169, 166–179 (2015).

  37. 37.

    Sewelam, N. et al. Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis. PLoS ONE 8, e70289 (2013).

  38. 38.

    Cominelli, E. et al. A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Curr. Biol. 15, 1196–1200 (2005).

  39. 39.

    Wang, W. H. et al. The reduced state of the plastoquinone pool is required for chloroplast-mediated stomatal closure in response to calcium stimulation. Plant J. 86, 132–144 (2016).

  40. 40.

    Frank, J. et al. Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis. New Phytol. 221, 866–880 (2019).

  41. 41.

    Schneider, A. et al. The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis. Plant Cell 28, 892–910 (2016).

  42. 42.

    Patron, M. et al. MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity. Mol. Cell 53, 726–737 (2014).

  43. 43.

    Raffaello, A. et al. The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. EMBO J. 32, 2362–2376 (2013).

  44. 44.

    Fadouloglou, V. E., Kokkinidis, M. & Glykos, N. M. Determination of protein oligomerization state: two approaches based on glutaraldehyde crosslinking. Anal. Biochem. 373, 404–406 (2008).

  45. 45.

    Maruyama, K., Mikawa, T. & Ebashi, S. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J. Biochem. 95, 511–519 (1984).

  46. 46.

    Wagner, S. et al. The EF-hand Ca2+ binding protein MICU choreographs mitochondrial Ca2+ dynamics in Arabidopsis. Plant cell 27, 3190–3212 (2015).

  47. 47.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

  48. 48.

    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).

  49. 49.

    Seigneurin-Berny, D., Salvi, D., Dorne, A. J., Joyard, J. & Rolland, N. Percoll-purified and photosynthetically active chloroplasts from Arabidopsis thaliana leaves. Plant Physiol. Biochem. 46, 951–955 (2008).

  50. 50.

    Brini, M. et al. Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. J. Biol. Chem. 270, 9896–9903 (1995).

  51. 51.

    Ottolini, D., Cali, T. & Brini, M. Methods to measure intracellular Ca2+ fluxes with organelle-targeted aequorin-based probes. Methods Enzymol. 543, 21–45 (2014).

  52. 52.

    Flury, P., Klauser, D., Schulze, B., Boller, T. & Bartels, S. The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst. Plant Physiol. 161, 2023–2035 (2013).

  53. 53.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2\({-\mathrm \Delta\mathrm \Delta}c_T\) method. Methods 25, 402–408 (2001).

Download references


The authors thank A. Weber, D. Leister, A. Costa, G. Finazzi, W. Martin and F. Lo Schiavo for useful discussions. We thank Human Frontiers Science Program (HFSP RG0052 to I.S.), the University of Padova (PRID 2018 prot. BIRD180317 to L.N. and STARS (Supporting Talents in Research project) to L.Carraretto), the MIUR (FFABR 2017 to E.F.), the EU within the Marie-Curie ITN CALIPSO (FP7, Project no. 607607 to U.C.V.) for financial support.

Author information

I.S., L.N., L.Cendron, E.F. and E.T. designed experiments; E.T., L.Carraretto, R.M., E.C., M.V., M.F., L.M. and S.D.B. performed experiments; I.S., L.N., L.Cendron, L.Carraretto, E.F., E.T., T.C. and U.C.V. analysed data; I.S., L.N., L.Cendron, E.F. and E.T. wrote the manuscript. I.S., L.N., E.F., U.C.V. and L.Carraretto acquired funding.

Correspondence to Elide Formentin or Lorella Navazio or Ildiko Szabo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Journal peer review information: Nature Plants thanks Jose Feijo, Simon Stael and Tou Cheu Xiong for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–17, Supplementary Video legends, Supplementary Table 1, Supplementary Materials and Methods, and Supplementary References.

Reporting Summary

Supplementary Video 1

Plants stably expressing mitochondrial β-F1-ATPase-EGFP (green) that have been transformed with cMCU::tdTomato (red).

Supplementary Video 2

cMCU::EGFP in epidermal cells stained with TMRM, a dye that accumulates in the mitochondria in a membrane potential-dependent manner

Supplementary Video 3

cMCU::EGFP in epidermal cells.

Supplementary Video 4

cMCU::EGFP in guard cells stained with TMRM (accumulates in mitochondria).

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Teardo, E., Carraretto, L., Moscatiello, R. et al. A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis. Nat. Plants 5, 581–588 (2019) doi:10.1038/s41477-019-0434-8

Download citation

Further reading