Plant Molecular Biology

, 77:145 | Cite as

Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1

  • Hala Badr Khalil
  • Zhejun Wang
  • Justin A. Wright
  • Alexandra Ralevski
  • Ariel O. Donayo
  • Patrick J. Gulick


The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca2+ signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein–protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca2+ levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca2+-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.


Heterotrimeric G protein alpha subunit Gα protein Calcium-binding protein Signal transduction GTPase-activating protein Phosphoinositide-specific phospholipase Protein–protein interaction 



This work is supported by grants from the Natural Science and Engineering Research Council of Canada, and the Agricultural Bioproducts Innovation Program of Agriculture and Agri-Food Canada. We thank Alan Jones, North Carolina State University, for kindly providing clones and vectors for control expression and protein–protein interaction. We thank Hugo Zheng, McGill University, for providing control clones for protein–protein interaction. The sequences of the T. aestivum genes described in the manuscript were deposited in GenBank with the following accession numbers: Ga3, HQ020506; PI-PLC1, HM754654; PI-PLC2, HM754653; Clo3, HQ020505.

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  1. Assmann SM (2005) G proteins go green: a plant G protein signaling FAQ sheet. Science 310:71–73PubMedCrossRefGoogle Scholar
  2. Berridge MJ (1993) Inositol trisphosphate and calcium signalling. Nature 361:315–325PubMedCrossRefGoogle Scholar
  3. Bisht NC, Jez JM, Pandey S (2011) An elaborate heterotrimeric G protein family from soybean expands the diversity of plant G-protein networks. New Phytol 190:35–48CrossRefGoogle Scholar
  4. Blumward E, Aharon GS, Lam BCH (1998) Early signal transduction pathways in plant-pathogen interactions. Trends Plant Sci 3:342–346CrossRefGoogle Scholar
  5. Chen JG (2008) Heterotrimeric G-protein signaling in Arabidopsis: puzzling G-protein-coupled receptor. Plant Signal Behav 3:1042–1045PubMedCrossRefGoogle Scholar
  6. Chen JG, Gao Y, Jones AM (2006) Differential roles of arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots. Plant Physiol 141:887–897PubMedCrossRefGoogle Scholar
  7. Cheong YH, Sung SJ, Kim BG, Pandey GK, Cho JS, Kim KN, Luan S (2010) Constitutive overexpression of the calcium sensor CBL5 confers osmotic or drought stress tolerance in Arabidopsis. Molec Cell 29:159–165CrossRefGoogle Scholar
  8. Claros MG, Von-Heijne G (1994) TopPred II, an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686PubMedGoogle Scholar
  9. Coonrod SA, Naaby-Hansen S, Shetty J, Shibahara H, Chen M, White JM, Herr JC (1999) Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion. Dev Biol 207:334–349PubMedCrossRefGoogle Scholar
  10. Dodd AN, Gardner MG, Hotta CT, Hubbard KE, Dalchau N, Love J, Assie J, Robertson FC, Jakobsen MK, Gonçalves J, Sanders D, Webb AAR (2007) The Arabidopsis Circadian Clock incorporates a cADPR-based feedback loop. Science 318:1789–1792PubMedCrossRefGoogle Scholar
  11. El-Maarouf H, Lameta A, Gareil M, Zuily-Fodil Y, Pham-Thi A (2001) Cloning and expression under drought of cDNAs coding for two PI-PLCs in cowpea leaves. Plant Physiol Biochem 39:167–172CrossRefGoogle Scholar
  12. Fan LM, Zhang W, Chen JG, Taylor JP, Jones AM, Assmann SM (2008) Abscisic acid regulation of guard-cell K+ and anion channels in G beta- and RGS-deficient Arabidopsis lines. Proc Natl Acad Sci USA 105:8476–8481PubMedCrossRefGoogle Scholar
  13. Fujita M, Fujita Y, Maruyama K, Seki M, Hirratsu K, Ohme-Takagi M, Tran LSP, Yamaguchi-Shinozaki K, Shinozaki K (2004) A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J 39:863–876PubMedCrossRefGoogle Scholar
  14. Grigston JC, Osuna D, Scheible WR, Liu C, Stitt M, Jones AM (2008) d-Glucose sensing by a plasma membrane regulator of G signaling protein, AtRGS1. FEBS Lett 582:3577–3584PubMedCrossRefGoogle Scholar
  15. Gulick PJ, Drouin S, Yu Z, Danyluk J (2005) Transcriptome comparison of winter and spring wheat responding to low temperature. Genome 48:913–923PubMedCrossRefGoogle Scholar
  16. Hamm HE (1998) The many faces of G protein signaling. J Biol Chem 273:669–672PubMedCrossRefGoogle Scholar
  17. Heinze M, Steighardt J, Gesell A, Schwartze W, Roos W (2007) Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica. Plant J 52:1041–1051PubMedCrossRefGoogle Scholar
  18. Hernandez-Pinzon I, Pateland K, Murphy DJ (2001) The Brassica napus calcium-binding protein, caleosin, has distinct endoplasmic reticulum- and lipid body-associated isoforms. Plant Physiol Biochem 39:615–622CrossRefGoogle Scholar
  19. Hossain MS, Koba T, Harada K (2003) Cloning and characterization of two full-length cDNAs, TaGA1 and TaGA2, encoding G-protein alpha subunits expressed differentially in wheat genome. Genes Genet Syst 78:127–138PubMedCrossRefGoogle Scholar
  20. Houde M, Belcaid M, Ouellet F, Danyluk J, Monroy AF, Dryanova A, Gulick P, Bergeron A, Laroche A, Links MG, MacCarthy L, Crosby WL, Sarhan F (2006) Wheat EST resources for functional genomics of abiotic stress. BMC Genom 7:149CrossRefGoogle Scholar
  21. Hu CD, Kerppola TK (2005) Direct visualization of protein interactions in living cells using bimolecular fluorescence complementation analysis. In: Adams P, Golemis E (eds) Protein–protein interactions, vol 34. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 1–20Google Scholar
  22. Huang CH, Crain RC (2009) Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton. Planta 230:925–933PubMedCrossRefGoogle Scholar
  23. Huang J, Taylor JP, Chen JG, Uhrig JF, Schnell DJ, Nakagawa T, Korth KL, Jones AM (2006) The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. Plant Cell 18:1226–1238PubMedCrossRefGoogle Scholar
  24. Johnston CA, Taylor JP, Gao Y, Kimple AJ, Grigston JC, Chen JG, Siderovski DP, Jones AM, Willard FS (2007) GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc Natl Acad Sci USA 104:17317–17322PubMedCrossRefGoogle Scholar
  25. Jones AM, Assmann SM (2004) Plants: the latest model system for G-protein research. EMBO Rep 5:572–578PubMedCrossRefGoogle Scholar
  26. Jones JC, Temple BR, Jones AM, Dohlman HG (2011) Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana. J Biol Chem 286:13143–13150PubMedCrossRefGoogle Scholar
  27. Kanuru M, Samuel JJ, Balivada LM, Aradhyam GK (2009) Ion-binding properties of Calnuc, Ca2+ versus Mg2+ Calnuc adopts additional and unusual Ca2+-binding sites upon interaction with G-protein. FEBS J 276:2529–2546PubMedCrossRefGoogle Scholar
  28. Kim Y, Jee K, Myoung L, Ho J, Young B, Byung H, Inhwan H, Woo K (2004) The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinsitide specific phospholipase C whose expression is induced by abiotic stress in mung bean Vigna radiate L. FEBS Lett 556:127–136PubMedCrossRefGoogle Scholar
  29. Kudla J, Batisti O, Hashimoto K (2010) Calcium signals: the lead currency of plant information processing. Plant Cell 22:541–563PubMedCrossRefGoogle Scholar
  30. Lapik YR, Kaufman LS (2003) The Arabidopsis cupin domain protein AtPirin 1 interacts with the G protein α-subunit GPA1 and regulates seed germination and early seedling development. Plant Cell 15:1578–1590PubMedCrossRefGoogle Scholar
  31. Mamillapalli R, Wysolmerski J (2010) The calcium-sensing receptor couples to Galpha(s) and regulates PTHrP and ACTH secretion in pituitary cells. J Endocrinol 204:287–297PubMedCrossRefGoogle Scholar
  32. Marrari Y, Crouthamel M, Irannejad R, Wedegaertner PB (2007) Assembly and trafficking of heterotrimeric G proteins. Biochemistry 46:7665–7677PubMedCrossRefGoogle Scholar
  33. Misra S, Yuliang W, Gayatri V, Sopory KS, Tuteja N (2007) Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C. Plant J 51:656–669PubMedCrossRefGoogle Scholar
  34. Monroy AF, Dryanova A, Malette B, Oren DH, Ridha Farajalla M, Liu W, Danyluk J, Ubayasena LWC, Kane K, Scoles GS, Sarhan F, Gulick PJ (2007) Regulatory gene candidates and gene expression analysis of cold acclimation in winter and spring wheat. Plant Mol Biol 64:409–423PubMedCrossRefGoogle Scholar
  35. Munnik T, Irvine RF, Musgrave A (1998) Phospholipid signalling in plants. Biochem Biophys Acta 1389:222–272PubMedGoogle Scholar
  36. Nakamura K, Sano H (2009) A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants. Plant Signal Behav 4:26–29PubMedCrossRefGoogle Scholar
  37. Nalefski EA, Falke JJ (1996) The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 5:2375–2390PubMedCrossRefGoogle Scholar
  38. Nelson KB, Xue C, Andreas N (2007) A multicolored set of in vivo organelle markers for co-localization studied in Arabidopsis and other plants. Plant J 51:1126–1136PubMedCrossRefGoogle Scholar
  39. Nilson SE, Assmann SM (2010) The alpha-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. Plant Physiol 152:2067–2077PubMedCrossRefGoogle Scholar
  40. Okamoto H, Göbel C, Capper RG, Saunders N, Feussner I, Knight MR (2009) The alpha-subunit of the heterotrimeric G-protein affects jasmonate responses in Arabidopsis thaliana. J Exp Bot 60:1991–2003PubMedCrossRefGoogle Scholar
  41. Pandey S, Assmann SM (2004) The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell 16:1616–1632PubMedCrossRefGoogle Scholar
  42. Pandey S, Chen JG, Jones AM, Assmann SM (2006) G-protein complex mutants are hypersensitive to abscisic acid regulation of germination and postgermination development. Plant Physiol 141:234–256CrossRefGoogle Scholar
  43. Pandey S, Nelson DC, Assmann SM (2009) Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell 136:136–148PubMedCrossRefGoogle Scholar
  44. Pandey S, Wang RS, Wilson L, Li S, Zhao Z, Gookin TE, Assmann SM, Albert R (2010) Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action. Mol Syst Biol 6:372–402PubMedCrossRefGoogle Scholar
  45. Partridge M, Murphy D (2009) Roles of a membrane-bound caleosin and putative peroxygenase in biotic and abiotic stress responses in Arabidopsis. Plant Physiol Biochem 47:796–806PubMedCrossRefGoogle Scholar
  46. Ritche S, Gilroy S (2000) Abscisic acid stimulation of phospholipase D in the barley aleurone is G-protein-mediated and localized to the plasma membrane. Plant Physiol 124:693–702CrossRefGoogle Scholar
  47. Song F, Goodman MR (2002) Molecular cloning and characterization of a rice phosphoinositide-specific phospholipase C gene, OsPI-PLC1, that is activated in systemic acquired resistance. Physiol Mol Plant Pathol 61:31–40Google Scholar
  48. Sprang SR (1997) G protein mechanisms: insights form structural analysis. Annu Rev Biochem 66:639–678PubMedCrossRefGoogle Scholar
  49. Steffens B, Sauter M (2010) G proteins as regulators in ethylene-mediated hypoxia signaling. Plant Signal Behav 5:375–378PubMedCrossRefGoogle Scholar
  50. Takahashi S, Katagiri T, Yamaguchi-Shinozaki K, Shinozaki K (2000) An Arabidopsis gene encoding a Ca2+-binding protein is induced by abscisic acid during dehydration. Plant Cell Physiol 41:898–903PubMedCrossRefGoogle Scholar
  51. Tardif G, Kane NA, Adam H, Labrie L, Major G, Gulick P, Sarhan F, Laliberte JF (2007) Interaction network of proteins associated with abiotic stress response and development in wheat. Plant Mol Biol 63:703–718PubMedCrossRefGoogle Scholar
  52. Thole JM, Nielsen E (2008) Phosphoinositides in plants: novel functions in membrane trafficking. Curr Opin Plant Biol 11:620–631PubMedCrossRefGoogle Scholar
  53. Todorova R (2009) In vitro interaction between the N-terminus of the Ewing’s sarcoma protein and the subunit of RNA polymerase II hsRPB7. Mol Biol Rep 36:1269–1274PubMedCrossRefGoogle Scholar
  54. Tosetti P, Pathak N, Jacob MH, Dunlap K (2003) RGS3 mediates a calcium-dependent termination of G protein signaling in sensory neurons. Proc Natl Acad Sci USA 100:7337–7342PubMedCrossRefGoogle Scholar
  55. Tuteja N, Sopory SK (2008) Chemical signaling under abiotic stress environment in plants. Plant Signal Behav 3:525–536PubMedCrossRefGoogle Scholar
  56. Ullah H, Chen JG, Temple B, Boyes DC, Alonso JM, Davis JM, Ecker JR, Jones AM (2003) The β-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. Plant Cell 15:393–409PubMedCrossRefGoogle Scholar
  57. Voinnet O, Rivas S, Mestre P, Baulcombe DC (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956PubMedCrossRefGoogle Scholar
  58. Vossen JH, Abd-El-Haliem A, Fradin EF, Berg GC, Ekengren SK, Meijer HJ, Seifi A, Bai Y, Have A, Munnik T, Thomma BP, Joosten MH (2010) Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance. Plant J 62:224–239PubMedCrossRefGoogle Scholar
  59. Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Hater K, Kudla J (2004) Visualization of protein interaction in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438PubMedCrossRefGoogle Scholar
  60. Wang XQ, Ullah H, Jones AM, Assmann SM (2001) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science 292:2070–2072PubMedCrossRefGoogle Scholar
  61. Wang CR, Yang AF, Yue GD, Gao Q, Yin HY, Zhang JR (2008) Enhanced expression of phospholipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize. Planta 227:1127–1140PubMedCrossRefGoogle Scholar
  62. Warpeha KM, Lateef SS, Lapik Y, Anderson M, Lee BS, Kaufman LS (2006) G-protein-coupled receptor 1, G-protein G alpha-subunit 1, and prephenate dehydratase 1 are required for blue light-induced production of phenylalanine in etiolated Arabidopsis. Plant Physiol 140:844–855PubMedCrossRefGoogle Scholar
  63. Weiss CA, White E, Huang H, Ma H (1997) The G protein α subunit (GPα1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower. FEBS Lett 407:361–367PubMedCrossRefGoogle Scholar
  64. Willard FS, Siderovski DP (2004) Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G-protein signaling-1. Methods Enzymol 389:320–338PubMedCrossRefGoogle Scholar
  65. Xu J, Tian YS, Peng RH, Xiong AS, Zhu B, Jin XF, Gao F, Fu XY, Hou XL, Yao QH (2010) AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis. Planta 231:1251–1260PubMedCrossRefGoogle Scholar
  66. Zhai S, Zhenhua S, Aifang Y, Zhang J (2005) Characterization of a novel phosphoinositide-specific phospholipase C from Zea mays and its expression in Escherichia coli. Biotech Lett 27:799–804CrossRefGoogle Scholar
  67. Zhao J, Wang X (2004) Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J Biol Chem 279:1794–1800PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Hala Badr Khalil
    • 1
  • Zhejun Wang
    • 1
  • Justin A. Wright
    • 1
  • Alexandra Ralevski
    • 1
  • Ariel O. Donayo
    • 1
    • 2
  • Patrick J. Gulick
    • 1
  1. 1.Department of BiologyConcordia UniversityMontrealCanada
  2. 2.Department of Biochemistry and Goodman Cancer Research CentreMcGill UniversityMontrealCanada

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