Journal of Plant Research

, Volume 124, Issue 3, pp 415–424 | Cite as

Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation

  • Takamitsu Kurusu
  • Haruyasu Hamada
  • Yoshimi Sugiyama
  • Toshikazu Yagala
  • Yasuhiro Kadota
  • Takuya Furuichi
  • Teruyuki Hayashi
  • Kenji Umemura
  • Setsuko Komatsu
  • Akio Miyao
  • Hirohiko Hirochika
  • Kazuyuki KuchitsuEmail author
Regular Paper


Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca2+ concentration ([Ca2+]cyt) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca2+ mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca2+]cyt, production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca2+ transients mainly due to plasma membrane Ca2+ influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca2+]cyt elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca2+ mobilization and may play a crucial role in temporal regulation of [Ca2+]cyt signature.


Calcium ion Calyculin A Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) Protein phosphorylation Reactive oxygen species 



We would like to thank Prof. Dr. Naoto Shibuya of Meiji University for gift of N-cetylchitooligosaccharides and suggestions, Prof. Dr. Kenzo Nakamura for the pIG121-Hm plasmid, Prof. Dr. Ko Shimamoto for suggestions, and Mr. Jumpei Hamada, Mr. Yohei Iwasaki, and Mr. Hiroshi Nokajima for technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (21200067 to T.K.), for Exploratory Research (21658118 to K.K.) and for Young Scientists (B) (21780041 to T.K.), and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated research project for plants, insects, and animals using genome technology MP-2134 to K.K.).

Supplementary material

10265_2010_388_MOESM1_ESM.tif (814 kb)
Supplemental figure S1 RT-PCR analysis of the apoaequorin transformant cell lines. Rice calli (Oryza sativa cultivar Nipponbare) were transformed with apoaequorin gene targetted to the cytoplasm. The gene expression was monitored by RT-PCR analysis. First-strand cDNA was synthesized from total RNA extracted from each cell line and amplified by RT-PCR as described in Materials and methods. Ubiquitin cDNA was used as a control. PCR products were analyzed by agarose gel electrophoresis. (TIFF 814 kb)
10265_2010_388_MOESM2_ESM.tif (491 kb)
Supplemental figure S2 The effect of several inhibitors on the N-acetylchitooligosaccharides-induced [Ca2+]cyt changes. Neomycin (500 μM), U-73122 (1 μM), and LaCl3 (5 mM) were added to the cells 15 min prior to the MAMP treatment (10 μM). Average values and SE of five independent experiments are shown. * P<0.05, ** P<0.01, significant difference as compared with the control according to unpaired Student’s t test. (TIFF 491 kb)
10265_2010_388_MOESM3_ESM.tif (769 kb)
Supplemental figure S3 Ionomycin-induced changes in [Ca2+]cyt in rice (Oryza sativa) cells. Data are representatives of five experiments with similar results. rlu, relative luminescence unit. (TIFF 768 kb)
10265_2010_388_MOESM4_ESM.tif (869 kb)
Supplemental figure S4 The Effect of a CaM antagonist, W7, on N-acetylchitooligosaccharides-induced [Ca2+]cyt changes. W7 (100 μM) were added to the rice (Oryza sativa) cells 15 min prior to the MAMP treatment (10 μM). Data are representatives of three experiments. rlu, relative luminescence unit. (TIFF 868 kb)


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Copyright information

© The Botanical Society of Japan and Springer 2010

Authors and Affiliations

  • Takamitsu Kurusu
    • 1
  • Haruyasu Hamada
    • 1
  • Yoshimi Sugiyama
    • 1
  • Toshikazu Yagala
    • 1
  • Yasuhiro Kadota
    • 1
    • 6
  • Takuya Furuichi
    • 2
  • Teruyuki Hayashi
    • 1
    • 7
  • Kenji Umemura
    • 3
  • Setsuko Komatsu
    • 4
  • Akio Miyao
    • 5
  • Hirohiko Hirochika
    • 5
  • Kazuyuki Kuchitsu
    • 1
    Email author
  1. 1.Department of Applied Biological ScienceTokyo University of ScienceNodaJapan
  2. 2.Institute of Plant Science and ResourcesOkayama UniversityKurashikiJapan
  3. 3.Agricultural and Veterinary Research LaboratoriesMeiji Seika Kaisha LtdYokohamaJapan
  4. 4.National Institute of Crop ScienceTsukubaJapan
  5. 5.Division of Genome and Biodiversity ResearchNational Institute of Agrobiological SciencesTsukubaJapan
  6. 6.The Sainsbury LaboratoryNorwich Research ParkNorwichUK
  7. 7.Division of Plant SciencesNational Institute of Agrobiological SciencesTsukubaJapan

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