Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Calcium signalling mediates self-incompatibility response in the Brassicaceae

An Erratum to this article was published on 03 September 2015


Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca2+ influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca2+ ([Ca2+]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca2+ increase, which was inhibited by D-(−)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca2+]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca2+ increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca2+ influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Dual pollination assay using self-incompatible A. thaliana.
Figure 2: Dynamics of [Ca2+]cyt in YC3.60pm-expressing papilla cells following self- and cross-pollination.
Figure 3: [Ca2+]cyt dynamics in papilla cell protoplasts treated with SP11/SCR.
Figure 4: A glutamate receptor antagonist AP-5 interferes with self-incompatibility response.
Figure 5: Glutamate receptor-like channel nonsense mutants are compromised in Ca2+ increase on self-incompatibility response.


  1. 1

    Takayama, S. & Isogai, A. Self-incompatibility in plants. Annu. Rev. Plant Biol. 56, 467–489 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Takayama, S. et al. Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature 413, 534–538 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Iwano, M. et al. Actin dynamics in papilla cells of Brassica rapa during self- and cross-pollination. Plant Physiol. 144, 72–81 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Samuel, M. A. et al. Proteomic analysis of Brassica stigmatic proteins following the self-incompatibility reaction reveals a role for microtubule dynamics during pollen responses. Mol. Cell Proteomics 10, M111.011338 (2011).

    Article  Google Scholar 

  5. 5

    Murase, K. et al. A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science 303, 1516–1519 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Kakita, M. et al. Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa. Plant Cell 19, 3961–3973 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Takada, Y. et al. Involvement of MLPK pathway in intraspecies unilateral incompatibility regulated by a single locus with stigma and pollen factors. G3 Bethesda 3, 719–726 (2013).

    CAS  Article  Google Scholar 

  8. 8

    Kitashiba, H., Liu, P., Nishio, T., Nasrallah, J. B. & Nasrallah, M. E. Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 108, 18173–18178 (2011).

    CAS  Article  Google Scholar 

  9. 9

    Gu, T. et al. Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proc. Natl Acad. Sci. USA 95, 382–387 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Stone, S. L., Arnoldo, M. & Goring, D. R. A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science 286, 1729–1731 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Stone, S. L., Anderson, E. M., Mullen, R. T. & Goring, D. R. ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell 15, 885–898 (2003).

    Article  Google Scholar 

  12. 12

    Samuel, M. A. et al. Cellular pathways regulating responses to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the exocyst complex. Plant Cell 21, 2655–2671 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Hsu, S. C., TerBush, D., Abraham, M. & Guo, W. The exocyst complex in polarized exocytosis. Int. Rev. Cytol. 233, 243–265 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Indriolo, E., Tharmapalan, P., Wright, S. I. & Goring, D. R. The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection. Plant Cell 24, 4607–4620 (2012).

    CAS  Article  Google Scholar 

  15. 15

    Indriolo, E., Safavian, D. & Goring, D. R. The ARC1 E3 ligase promotes two different self-pollen avoidance traits in Arabidopsis. Plant Cell 26, 1525–1543 (2014).

    CAS  Article  Google Scholar 

  16. 16

    Nasrallah, J. B. & Nasrallah, M. E. Robust self-incompatibility in the absence of a functional ARC1 gene in Arabidopsis thaliana. Plant Cell 26, 3838–3841 (2014).

    CAS  Article  Google Scholar 

  17. 17

    Goring, D. R., Indriolo, E. & Samuel, M. A. The ARC1 E3 ligase promotes a strong and stable self-incompatibility response in Arabidopsis species: response to the Nasrallah and Nasrallah commentary. Plant Cell 26, 3842–3846 (2014).

    CAS  Article  Google Scholar 

  18. 18

    Dearnaley, J. D., Levina, N. N., Lew, R. R., Heath, I. B. & Goring, D. R. Interrelationships between cytoplasmic Ca2+ peaks, pollen hydration and plasma membrane conductances during compatible and incompatible pollinations of Brassica napus papillae. Plant Cell Physiol. 38, 985–999 (1997).

    CAS  Article  Google Scholar 

  19. 19

    Sherman-Broyles, S. et al. S locus genes and the evolution of self-fertility in Arabidopsis thaliana. Plant Cell 19, 94–106 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Tsuchimatsu, T. et al. Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene. Nature 464, 1342–1346 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Dwyer, K. G. et al. Molecular characterization and evolution of self-incompatibility genes in Arabidopsis thaliana: the case of the Sc haplotype. Genetics 193, 985–994 (2013).

    CAS  Article  Google Scholar 

  22. 22

    Nasrallah, M. E., Liu, P. & Nasrallah, J. B. Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata . Science 297, 247–249 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Nasrallah, M. E., Liu, P., Sherman-Broyles, S., Boggs, N. A. & Nasrallah, J. B. Natural variation in expression of self-incompatibility in Arabidopsis thaliana: implications for the evolution of selfing. Proc. Natl Acad. Sci. USA 101, 16070–16074 (2004).

    CAS  Article  Google Scholar 

  24. 24

    Goring, D. R. & Rothstein, S. J. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. Plant Cell 4, 1273–1281 (1992).

    CAS  Article  Google Scholar 

  25. 25

    Tantikanjana, T., Rizvi, N., Nasrallah, M. E. & Nasrallah, J. B. A dual role for the S-locus receptor kinase in self-incompatibility and pistil development revealed by an Arabidopsis rdr6 mutation. Plant Cell 21, 2642–2654 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Sarker, R. H., Elleman, C. J. & Dickinson, H. G. Control of pollen hydration in Brassica requires continued protein synthesis, and glycosylation in necessary for intraspecific incompatibility. Proc. Natl Acad. Sci. USA 85, 4340–4344 (1988).

    CAS  Article  Google Scholar 

  27. 27

    Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl Acad. Sci. USA 101, 10554–10559 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Iwano, M. et al. Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol. 136, 3562–3571 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Shimosato, H. et al. Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in Brassica self-incompatibility. Plant Cell 19, 107–117 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Gee, K. R. et al. Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium 27, 97–106 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Knight, H., Trewavas, A. J. & Knight, M. R. Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J. 12, 1067–1078 (1997).

    CAS  Article  Google Scholar 

  32. 32

    Michard, E. et al. Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine. Science 332, 434–437 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Parre, E. et al. Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis. Plant Physiol. 144, 503–512 (2007).

    CAS  Article  Google Scholar 

  34. 34

    White, P. J. Calcium channels in higher plants. Biochim. Biophys. Acta 1465, 171–189 (2000).

    CAS  Article  Google Scholar 

  35. 35

    Schuh, K. et al. Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility. J. Biol. Chem. 279, 28220–28226 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Okuda, S. et al. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458, 357–361 (2009).

    CAS  Article  Google Scholar 

  37. 37

    Denninger, P. et al. Male–female communication triggers calcium signatures during fertilization in Arabidopsis. Nature Commun. 5, 4645 (2014).

    CAS  Article  Google Scholar 

  38. 38

    Kerhoas, C., Knox, R. B. & Dumas, C. Specificity of the callose response in stigmas of Brassica. Ann. Bot. 52, 597–602 (1983).

    Article  Google Scholar 

  39. 39

    Kauss, H. Callose biosynthesis as a Ca2+-regulated process and possible relations to the induction of other metabolic changes. J. Cell Sci. Suppl. 2, 89–103 (1985).

    Article  Google Scholar 

  40. 40

    Price, M. B., Jelesko, J. & Okumoto, S. Glutamate receptor homologs in plants: functions and evolutionary origins. Front. Plant Sci. 3, 1–10 (2012).

    Article  Google Scholar 

  41. 41

    Roy, S. J. et al. Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana. Plant, Cell & Environ. 31, 861–871 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Iwano, M. et al. A pollen coat–inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae. Plant Cell 26, 636–649 (2014).

    CAS  Article  Google Scholar 

  43. 43

    Iwano, M. & Takayama, S. Self/non-self discrimination in angiosperm self-incompatibility. Curr. Opin. Plant Biol. 15, 78–83 (2011).

    Article  Google Scholar 

  44. 44

    Wheeler, M. J. et al. Identification of the pollen self-incompatibility determinant in Papaver rhoeas. Nature 459, 992–995 (2009).

    CAS  Article  Google Scholar 

  45. 45

    Wheeler, M. J., Vatovec, S. & Franklin-Tong, V. E. The pollen S-determinant in Papaver: comparisons with known plant receptors and protein ligand partners. J. Exp. Bot. 61, 2015–2025 (2010).

    CAS  Article  Google Scholar 

  46. 46

    Harada, Y. et al. Mechanism of self-sterility in a hermaphroditic chordate. Science 320, 548–550 (2008).

    CAS  Article  Google Scholar 

  47. 47

    Saito, T., Shiba, K., Inaba, K., Yamada, L. & Sawada, H. Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis. Proc. Natl Acad. Sci. USA 109, 4158–4162 (2012).

    CAS  Article  Google Scholar 

  48. 48

    Iwano, M. et al. Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth. Plant Physiol. 150, 1322–1334 (2009).

    CAS  Article  Google Scholar 

  49. 49

    Shiba, H. et al. Alteration of the self-incompatibility phenotype in Brassica by transformation of the antisense SLG gene. Biosci. Biotechnol. Biochem. 64, 1016–1024 (2000).

    CAS  Article  Google Scholar 

Download references


We thank Y. Ryokume, M. Okamura, F. Yamamoto, and M. Matsumura-Kawashima for their technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (21112003 to M. Iwano; 23113002 to S.T.), Grants-in-Aid for Scientific Research (23570056, 26440145 to M. Iwano; 21248014, 25252021 to S.T.), and Grants-in-Aid for Creative Scientific Research (16GS0316 to A.I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); and by Grants-in-Aid for JSPS Research Fellow (12J08273 to K.I.) from the Japan Society for the Promotion of Science (JSPS).

Author information




M. Iwano, A.I. and S.T. initiated the project. M. Iwano, K.I., M.K., P.K.-N., T.N., A.M. and S.T. designed experiments. M. Iwano, K.I., H.A.-S., S.F., M. Igarashi, T.E., A.K., M.T., M.T., K.K. and H.S. performed the experiments. M. Iwano, K.I., S.F. and S.T. wrote the manuscript.

Corresponding author

Correspondence to Seiji Takayama.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Iwano, M., Ito, K., Fujii, S. et al. Calcium signalling mediates self-incompatibility response in the Brassicaceae. Nature Plants 1, 15128 (2015).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing