Plant Molecular Biology

, Volume 95, Issue 4–5, pp 463–479 | Cite as

Functional characterization of chloroplast-targeted RbgA GTPase in higher plants

  • Young Jeon
  • Hee-Kyung Ahn
  • Yong Won Kang
  • Hyun-Sook Pai


Key message

Plant RbgA GTPase is targeted to chloroplasts and co-fractionated with chloroplast ribosomes, and plays a role in chloroplast rRNA processing and/or ribosome biogenesis.


Ribosome Biogenesis GTPase A (RbgA) homologs are evolutionarily conserved GTPases that are widely distributed in both prokaryotes and eukaryotes. In this study, we investigated functions of chloroplast-targeted RbgA. Nicotiana benthamiana RbgA (NbRbgA) and Arabidopsis thaliana RbgA (AtRbgA) contained a conserved GTP-binding domain and a plant-specific C-terminal domain. NbRbgA and AtRbgA were mainly localized in chloroplasts, and possessed GTPase activity. Since Arabidopsis rbgA null mutants exhibited an embryonic lethal phenotype, virus-induced gene silencing (VIGS) of NbRbgA was performed in N. benthamiana. NbRbgA VIGS resulted in a leaf-yellowing phenotype caused by disrupted chloroplast development. NbRbgA was mainly co-fractionated with 50S/70S ribosomes and interacted with the chloroplast ribosomal proteins cpRPL6 and cpRPL35. NbRbgA deficiency lowered the levels of mature 23S and 16S rRNAs in chloroplasts and caused processing defects. Sucrose density gradient sedimentation revealed that NbRbgA-deficient chloroplasts contained reduced levels of mature 23S and 16S rRNAs and diverse plastid-encoded mRNAs in the polysomal fractions, suggesting decreased protein translation activity in the chloroplasts. Interestingly, NbRbgA protein was highly unstable under high light stress, suggesting its possible involvement in the control of chloroplast ribosome biogenesis under environmental stresses. Collectively, these results suggest a role for RbgA GTPase in chloroplast rRNA processing/ribosome biogenesis, affecting chloroplast protein translation in higher plants.


Chloroplast abnormality Nicotiana benthamiana Ribosomal RNA processing Ribosome association Virus-induced gene silencing 



This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development [Project Numbers PJ01118901 (Systems & Synthetic Agrobiotech Center) and PJ01114701 (Plant Molecular Breeding Center)] from the Rural Development Administration (to H.-S. Pai), and the Basic Science Research Program (Project Number 2016-11-1224) from the National Research Foundation of Republic of Korea (to Y. Jeon).

Author contributions

Y.J. performed all of the experiments with the help of H.-K.A and Y.W.K. Y.J. and H.-S.P. designed the experiments, discussed the results, and wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2017_664_MOESM1_ESM.pdf (442 kb)
Supplementary material 1 (PDF 441 KB)


  1. Achila D, Gulati M, Jain N, Britton RA (2012) Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis. J Biol Chem 287:8417–8423CrossRefPubMedPubMedCentralGoogle Scholar
  2. Adilakshmi T, Bellur DL, Woodson SA (2008) Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly. Nature 455:1268–1272CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ahmed T, Yin Z, Bhushan S (2016) Cryo-EM structure of the large subunit of the spinach chloroplast ribosome. Sci Rep 6:35793CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ahn CS, Han J-A, Lee H-S, Lee S, Pai H-S (2011) The PP2A regulatory subunit Tap46, a component of TOR signaling pathway, modulates growth and metabolism in plants. Plant Cell 23:185–209CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ahn CS, Cho HK, Lee D-H, Sim H-J, Kim S-G, Pai H-S (2016) Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW), and mechanisms of defective cell growth and proliferation caused by PeBoW deficiency in Arabidopsis. J Exp Bot 67:5217–5232CrossRefPubMedPubMedCentralGoogle Scholar
  6. Aro EM, Virgin I, Andersson B (1993) Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochim Biophys Acta 1143:113–134CrossRefPubMedGoogle Scholar
  7. Bang WY, Chen J, Jeong IS, Kim SW, Kim CW, Jung HS, Lee KH, Kweon HS, Yoko I, Shiina T, Bahk JD (2012) Functional characterization of ObgC in ribosome biogenesis during chloroplast development. Plant J 71:122–134CrossRefPubMedGoogle Scholar
  8. Bellaoui M, Keddie JS, Gruissem W (2003) DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development. Plant Mol Biol 53:531–543CrossRefPubMedGoogle Scholar
  9. Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316CrossRefPubMedGoogle Scholar
  10. Bollenbach TJ, Lange H, Gutierrez R, Erhardt M, Stern DB, Gagliardi D (2005) RNR1, a 3ʹ-5ʹ exoribonuclease belonging to the RNR superfamily, catalyzes 3ʹ maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana. Nucleic Acids Res 33:2751–2763CrossRefPubMedPubMedCentralGoogle Scholar
  11. Britton RA (2009) Role of GTPases in bacterial ribosome assembly. Annu Rev Microbiol 63:155–176CrossRefPubMedGoogle Scholar
  12. Caldon CE, March PE (2003) Function of the universally conserved bacterial GTPases. Curr Opin Microbiol 6:135–139CrossRefPubMedGoogle Scholar
  13. Chazotte B (2011) Labeling mitochondria with TMRM or TMRE. Cold Spring Harb Protoc 2011:895–897PubMedGoogle Scholar
  14. Cho HK, Ahn CS, Lee H-S, Kim J-K, Pai HS (2013) Pescadillo plays an essential role in plant cell growth and survival by modulating ribosome biogenesis. Plant J 76:393–405CrossRefPubMedGoogle Scholar
  15. Corrigan RM, Bellows LE, Wood A, Gründling A (2016) ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria. Proc Natl Acad Sci USA 113:E1710–E1719CrossRefPubMedPubMedCentralGoogle Scholar
  16. Dalebroux ZD, Swanson MS (2012) ppGpp: magic beyond RNA polymerase. Nature Rev Microbiol 10:203–212CrossRefGoogle Scholar
  17. Daviter T, Wieden HJ, Rodnina MV (2003) Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome. J Mol Biol 332:689–699CrossRefPubMedGoogle Scholar
  18. Edelman M, Mattoo AK (2008) D1-protein dynamics in photosystem II: the lingering enigma. Photosynth Res 98:609–620CrossRefPubMedGoogle Scholar
  19. Ellis RJ (1969) Chloroplast ribosomes: stereospecificity of inhibition by chloramphenicol. Science 163:477–478CrossRefPubMedGoogle Scholar
  20. Gulati M, Jain N, Anand B, Prakash B, Britton RA (2013) Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation. Nucleic Acids Res 41:3217–3227CrossRefPubMedPubMedCentralGoogle Scholar
  21. Gulati M, Jain N, Davis JH, Williamson JR, Britton RA (2014) Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly. PLoS Genet 10:e1004694CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hedges J, West M, Johnson AW (2005) Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p. EMBO J 24:567–579CrossRefPubMedPubMedCentralGoogle Scholar
  23. Herold M, Nierhaus KH (1987) Incorporation of six additional proteins to complete the assembly map of the 50S subunit from Escherichia coli ribosomes. J Biol Chem 262:8826–8833PubMedGoogle Scholar
  24. Hideg E, Kálai T, Hideg K, Vass I (2000) Do oxidative stress conditions impairing photosynthesis in the light manifest as photoinhibition? Philos Trans R Soc Lond B Biol Sci 355:1511–1516CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hill TA, Broadhvest J, Kuzoff RK, Gasser CS (2006) Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase. Genetics 174:707–718CrossRefPubMedPubMedCentralGoogle Scholar
  26. Im CH, Hwang SM, Son YS, Heo JB, Bang WY, Suwastika IN, Shiina T, Bahk JD (2011) Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants. J Biol Chem 286:8620–8632CrossRefPubMedPubMedCentralGoogle Scholar
  27. Inoue K, Alsina J, Chen J, Inouye M (2003) Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli. Mol Microbiol 48:1005–1016CrossRefPubMedGoogle Scholar
  28. Jeon Y, Ahn CS, Jung HJ, Kang H, Park GT, Choi Y, Hwang J, Pai HS (2014) DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants. J Exp Bot 65:117–130CrossRefPubMedGoogle Scholar
  29. Jeon Y, Park YJ, Cho HK, Jung HJ, Ahn TK, Kang H, Pai HS (2015) The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis. J Exp Bot 66:6297–6310CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jomaa A, Jain N, Davis JH, Williamson JR, Britton RA, Ortega J (2014) Functional domains of the 50S subunit mature late in the assembly process. Nucleic Acids Res 42:3419–3435CrossRefPubMedGoogle Scholar
  31. Kojima K, Motohashi K, Morota T, Oshita M, Hisabori T, Hayashi H, Nishiyama Y (2009) Regulation of translation by the redox state of elongation factor G in the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 284:18685–18691CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kössel H, Edwards K, Koch W, Langridge P, Schiefermayr E, Schwarz Z, Strittmatter G, Zenke G (1982) Structural and functional analysis of an rRNA operon and its flanking tRNA genes from Zea mays chloroplasts. Nucleic Acids Symp Ser 11:117–120Google Scholar
  33. Kristiansen KA, Jensen PE, Møller IM, Schulz A (2009) Monitoring reactive oxygen species formation and localisation in living cells by use of the fluorescent probe CM-H2DCFDA and confocal laser microscopy. Physiol Plant 136:369–383CrossRefPubMedGoogle Scholar
  34. Li Z, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and responding to excess light. Annu Rev Plant Biol 60:239–260CrossRefPubMedGoogle Scholar
  35. Li N, Chen Y, Guo Q, Zhang Y, Yuan Y, Ma C, Deng H, Lei J, Gao N (2013) Cryo-EM structures of the late-stage assembly intermediates of the bacterial 50S ribosomal subunit. Nucleic Acids Res 41:7073–7083CrossRefPubMedPubMedCentralGoogle Scholar
  36. Liu H, Lau E, Lam MP et al (2010) OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function. New Phytol 187:83–105CrossRefPubMedGoogle Scholar
  37. Manuell AL, Quispe J, Mayfield SP (2007) Structure of the chloroplast ribosome: novel domains for translation regulation. PLoS Biol 5:e209CrossRefPubMedPubMedCentralGoogle Scholar
  38. Matsuo Y, Morimoto T, Kuwano M, Loh PC, Oshima T, Ogasawara N (2006) The GTP-binding protein YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. J Biol Chem 281:8110–8117CrossRefPubMedGoogle Scholar
  39. Matsuo Y, Oshima T, Loh PC, Morimoto T, Ogasawara N (2007) Isolation and characterization of a dominant negative mutant of Bacillus subtilis GTP-binding protein, YlqF, essential for biogenesis and maintenance of the 50 S ribosomal subunit. J Biol Chem 282:25270–25277CrossRefPubMedGoogle Scholar
  40. Nishiyama Y, Allakhverdiev SI, Murata N (2006) A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. Biochim Biophys Acta 1757:742–749CrossRefPubMedGoogle Scholar
  41. Nishiyama Y, Allakhverdiev SI, Murata N (2011) Protein synthesis is the primary target of reactive oxygen species in the photoinhibition of photosystem II. Physiol Plant 142:35–46CrossRefPubMedGoogle Scholar
  42. Röhl R, Nierhaus KH (1982) Assembly map of the large subunit (50S) of Escherichia coli ribosomes. Proc Natl Acad Sci USA 79:729–733CrossRefPubMedPubMedCentralGoogle Scholar
  43. Romani I, Tadini L, Rossi F, Masiero S, Pribil M, Jahns P, Kater M, Leister D, Pesaresi P (2012) Versatile roles of Arabidopsis plastid ribosomal proteins in plant growth and development. Plant J 72:922–934CrossRefPubMedGoogle Scholar
  44. Sharma MR, Wilson DN, Datta PP, Barat C, Schluenzen F, Fucini P, Agrawal RK (2007) Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins. Proc Natl Acad Sci USA 104:19315–19320CrossRefPubMedPubMedCentralGoogle Scholar
  45. Shu CJ, Zhulin IB (2002) ANTAR: an RNA-binding domain in transcription antitermination regulatory proteins. Trends Biochem Sci 27:3–5CrossRefPubMedGoogle Scholar
  46. Stadtman ER (2006) Protein oxidation and aging. Free Radic Res 40:1250–1258CrossRefPubMedGoogle Scholar
  47. Strittmatter G, Kössel H (1984) Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays. Nucleic Acids Res 12:7633–7647CrossRefPubMedPubMedCentralGoogle Scholar
  48. Sykes MT, Williamson JR (2009) A complex assembly landscape for the 30S ribosomal subunit. Annu Rev Biophys 38:197–215CrossRefPubMedPubMedCentralGoogle Scholar
  49. Takahashi S, Murata N (2008) How do environmental stresses accelerate photoinhibition? Trends Plant Sci 13:178–182CrossRefPubMedGoogle Scholar
  50. Talkington MW, Siuzdak G, Williamson JR (2005) An assembly landscape for the 30S ribosomal subunit. Nature 438:628–632CrossRefPubMedPubMedCentralGoogle Scholar
  51. Tiller N, Bock R (2014) The translational apparatus of plastids and its role in plant development. Mol Plant 7:1105–1120CrossRefPubMedPubMedCentralGoogle Scholar
  52. Traub P, Nomura M (1968) Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc Natl Acad Sci USA 59:777–784CrossRefPubMedPubMedCentralGoogle Scholar
  53. Traub P, Nomura M (1969) Structure and function of Escherichia coli ribosomes. VI. Mechanism of assembly of 30 s ribosomes studied in vitro. J Mol Biol 40:391–413CrossRefPubMedGoogle Scholar
  54. Triantaphylidès C, Havaux M (2009) Singlet oxygen in plants: production, detoxification and signaling. Trends Plant Sci 14:219–228CrossRefPubMedGoogle Scholar
  55. Triantaphylidès C, Krischke M, Hoeberichts FA, Ksas B, Gresser G, Havaux M, Van Breusegem F, Mueller MJ (2008) Singlet oxygen is the major reactive oxygen species involved in photooxidative damage to plants. Plant Physiol 148:960–968CrossRefPubMedPubMedCentralGoogle Scholar
  56. Uicker WC, Schaefer L, Britton RA (2006) The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis. Mol Microbiol 59:528–540CrossRefPubMedGoogle Scholar
  57. Verstraeten N, Fauvart M, Versées W, Michiels J (2011) The universally conserved prokaryotic GTPases. Microbiol Mol Biol Rev 75:507–542CrossRefPubMedPubMedCentralGoogle Scholar
  58. Walter M, Chaban C, Schütze K et al (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438CrossRefPubMedGoogle Scholar
  59. Wang X, Gingrich DK, Deng Y, Hong Z (2012) A nucleostemin-like GTPase required for normal apical and floral meristem development in Arabidopsis. Mol Biol Cell 23:1446–1456CrossRefPubMedPubMedCentralGoogle Scholar
  60. Weis BL, Missbach S, Marzi J, Bohnsack MT, Schleiff E (2014) The 60S associated ribosome biogenesis factor LSG1-2 is required for 40S maturation in Arabidopsis thaliana. Plant J 280:1043–1056CrossRefGoogle Scholar
  61. Wekselman I, Davidovich C, Agmon I, Zimmerman E, Rozenberg H, Bashan A, Berisio R, Yonath A (2009) Ribosome’s mode of function: myths, facts and recent results. J Pept Sci 15:122–130CrossRefPubMedGoogle Scholar
  62. Woolford JL Jr, Baserga SJ (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195:643–681CrossRefPubMedPubMedCentralGoogle Scholar
  63. Yamaguchi K, Subramanian AR (2000) The plastid ribosomal proteins: identification of all the proteins in the 50S subunit of an organelle ribosome (chloroplast). J Biol Chem 275:28466–28482CrossRefPubMedGoogle Scholar
  64. Yamaguchi K, Subramanian AR (2003) Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit. Eur J Biochem 270:190–205CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of Systems BiologyYonsei UniversitySeoulSouth Korea
  2. 2.R&D Center, Morechem Co., Ltd.YonginSouth Korea

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