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Acta Agron Sin ›› 2013, Vol. 39 ›› Issue (11): 1952-1961.doi: 10.3724/SP.J.1006.2013.01952

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Characteristic and Function Analysis of a Copper Ion Binding Protein, AtBCB Interacting with G Protein α Subunit GPA1 in Arabidopsis thaliana

ZHANG Xiao-Hong1,XU Peng-Bo1,2,GUO Meng-Meng1,2,XU Zhao-Shi2,LI Lian-Cheng2,CHEN Ming2,*,MA You-Zhi2   

  1. 1 College of Life Sciences, Northwest A&F University, Yangling 712100, China; 2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / National Key Facility for Crop Gene Resources and Genetic Improvement, Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 100081
  • Received:2013-04-07 Revised:2013-06-09 Online:2013-11-12 Published:2013-08-01
  • Contact: 陈明, E-mail: chenming02@caas.cn; Tel: 010-82108750

Abstract:

Heterotrimeric G protein, including three subunits of α, β, and γ, is involved in a lot of signaling pathways in plants. It receives extracellular signals via G-protein coupled receptor (GPCR) and transmits them to the downstream effectors by the three subunits. Till now, the downstream effectors and signaling pathway related to G-protein complexes have been rarely reported. Furthermore, identifying more novel G protein effectors would be helpful to elucidate signaling pathway associated with the G protein complex. In order to find some novel effectors, G protein α subunit (GPA1) was used as bait to screen interaction protein in Arabidopsis by the split-ubiquitin screening system in this study. One of the GPA1-interacting proteins was identified as copper ion binding protein,AtBCB. The interaction betweenGPA1 and AtBCB was verified on cell membrane by BiFC (bimolecular fluorescence complementation). The expressions of GPA1 and AtBCB were confirmed to be induced by aluminium stress. To study the function of the two genes, we treated Arabidopsis mutant gpa1-4 and bcb, in which GPA1 and AtBCB were knocked out, with 100 µmol L–1 Al3+, respectively, and then measured MDA (malonaldehyde) content in roots. The results showed that MDA content in both mutant and WT (wild type) under normal condition was no significant difference, but when exposured to 100 µmol L–1 Al3+ the content in gpa1-4 was lower than that in WT (P<0.05), and it in bcb was higher than that in WT (P<0.01). Furthermore, the expression patterns of three responsive genes ofALMT1, ALS1 and ALS3 to aluminum toxicity were analyzed by Real-time PCR. The results showed that no matter the condition with or without aluminum stress, the expression of ALS1 and ALS3 appeared no significant difference in the mutants and WT. However, in the treatment with less than 100 µmol L–1 Al the expression level of ALMT1 ingpa1-4 was higher than that in WT, and the expression level of ALMT1 inbcbwas lower than that in WT. In short, GPA1 and AtBCB directly interact in the cell membrane, and regulate the expression of the downstream gene of ALMT1. In the tolerance process to aluminum stress in plants, GPA1 plays negative role but AtBCB has positive effect.

Key words: G protein, Split-ubiquitin system, Protein interaction, Aluminium ion stress, BiFC

[1]Chakravorty D, Trusov Y, Zhang W, Acharya B R, Sheahan M B, McCurdy D W, Assmann S M, Botella J R. An atypical heterotrimeric G-protein gamma-subunit is involved in guard cell K+-channel regulation and morphological development in Arabidopsis thaliana. Plant J, 2011, 67: 840–851



[2]Warpeha K M, Upadhyay S, Yeh J, Adamiak J, Hawkins S I, Lapik Y R, Anderson M B, Kaufman L S. The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis. Plant Physiol, 2007, 143: 1590–1600



[3]Botto J F, Ibarra S, Jones A M. The heterotrimeric G-protein complex modulates light sensitivity in Arabidopsis thaliana seed germination. Photochem Photobiol, 2009, 85: 949–954



[4]Fox A R, Soto G C, Jones A M, Casal J J, Muschietti J P, Mazzella M A. Cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis. Plant Mol Biol, 2012, 80: 315–324



[5]Wang X Q, Ullah H, Jones A M, Assmann S M. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science, 2001, 292: 2070–2072



[6]Mudgil Y, Uhrig J F, Zhou J, Temple B, Jiang K, Jones A M. Arabidopsis N-MYC downregulated-like 1, a positive regulator of auxin transport in a G protein-mediated pathway. Plant Cell, 2009, 21: 3591–3609



[7]Huang J, Taylor J P, Chen J G, Uhrig J F, Schnell D J, Nakagawa T, Korth K L, Jones A M. The plastid protein thylakoid formation 1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. Plant Cell, 2006, 18: 1226–1238



[8]Assmann S M, Fan L M, Zhang W, Chen J G, Taylor J P, Jones A M. Abscisic acid regulation of guard-cell K+ and anion channels in G beta- and RGS-deficient Arabidopsis lines. Proc Natl Acad Sci USA, 2008, 105: 8476–8481



[9]Llorente F, Blanco C A, Rodriguez C S, Jorda L, Molina A. ERECTA receptor-like kinase and heterotrimeric G protein from Arabidopsis are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina. Plant J, 2005, 43: 165–180



[10]Lapik Y R, Kaufman L S. The Arabidopsis cupin domain protein AtPirin1 interacts with the G protein alpha subunit GPA1 and regulates seed germination and early seedling development. Sci STKE, 2003, 15: 1578–1590



[11]Assmann S M, Pandey S. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell, 2004, 16: 1616–1632



[12]Zhao J, Wang X. Arabidopsis phospholipase D alpha 1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. Sci Signal, 2004, 279: 1794–1800



[13]Broder Y C, Katz S, Aronheim A. The ras recruitment system, a novel approach to the study of protein–protein interactions. Curr Biol, 1998, 8: 1121–1130



[14]Hubsman M, Yudkovsky G, Aronheim A. A novel approach for the identification of protein–protein interaction with integral membrane proteins. Nucl Acids Res, 2001, 29: e18



[15]Yuan G-L (苑国良). Characterizing Arabidopsis G Protein Interactors by Reverse Ras Recruitment System. PhD Disseratation of Shandong Agricultural University, 2009 (in Chinese with English abstract)



[16]Kaufman L S, Warpeha K M, Gibbons J, Carol A, Slusser J, Tree R, Durham W. Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings. Plant Cell Environ, 2008, 31: 1756–1770



[17]Friedman E J, Wang H X, Jiang K, Perovic I, Deshpande A, Pochapsky T C, Temple B R S, Hicks S N, Harden T K, Jones A M. Aci-reduction dioxygenase1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis. 2011, 286: 30107–30118



[18]Tsugama D, Liu H, Liu S, Takano T. Arabidopsis heterotrimeric G protein β subunit interacts with a plasma membrane 2C-type protein phosphatase, PP2C52. Biochimt Biophysica Acta (BBA)-Mol Cell Res, 2012: 2254–2260



[19]Stagljar I, Korostensky C, Johnsson N, te Heesen S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci USA, 1998, 95: 5187–5192



[20]Takumi S, Otani M, Shimada T. Effect of six promoter-intron combinations on transient reporter gene expression in einkorn, emmer and common wheat cells by particle bombardment. Plant Sci, 1994, 103: 161–166



[21]Li M(李敏), Yang S(杨双), Ruan Y-Y(阮燕晔), Fan J-J(樊金娟), Zhang L-J(张立军). PCR identification Arabidopsis T-DNA mutant of atsuc3. Plant Physiol Commun (植物生理学通讯), 2006, 42(1): 91–94 (in Chinese)



[22]Murphy A, Taiz L. A new vertical mesh transfer technique for metal-tolerance studies in Arabidopsis–ecotypic variation and copper-sensitive mutants. Plant Physiol, 1995, 108: 29–38



[23]Ono K, Yamamoto Y, Hachiya A, Matsumoto H. Synergistic inhibition of growth by aluminum and iron of tobacco (Nicotiana tabacum L.) cells in suspension culture. Plant Cell Physiol, 1995, 36: 115–125



[24]Van Gysel A, Van Montagu M, Inzé D. A negatively light-regulated gene from Arabidopsis thaliana encodes a protein showing high similarity to blue copper-binding proteins. Gene, 1993, 136: 79–85



[25]Richards K D, Schott E J, Sharma Y K, Davis K R, Gardner R C. Aluminum induces oxidative stress genes in Arabidopsis thaliana. Plant Physiol, 1998, 116: 409–418



[26]Ezaki B, Sasaki K, Matsumoto H, Nakashima S. Functions of two genes in aluminium (Al) stress resistance: repression of oxidative damage by the AtBCB gene and promotion of efflux of Al ions by the NtGDI1gene. J Exp Bot, 2005, 56: 2661–2671



[27]Kochian L V. Cellular Mechanisms of Aluminum Toxicity and Resistance in Plants. Annu Rev Plant Physiol Plant Mol Biol, 1995, 46: 237–260



[28]Ezaki B, Gardner R C, Ezaki Y, Matsumoto H. Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress. Plant Physiol, 2000, 122: 657–666



[29]Ezaki B, Katsuhara M, Kawamura M, Matsumoto H. Different mechanisms of four aluminum (Al)-resistant transgenes for Al toxicity in Arabidopsis. Plant Physiol, 2001, 127: 918–927



[30]Hoekenga O A, Maron L G, Piñeros M A, Cançado G M, Shaff J, Kobayashi Y, Ryan P R, Dong B, Delhaize E, Sasaki T. AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci USA, 2006, 103: 9738–9743



[31]Larsen P B, Geisler M J, Jones C A, Williams K M, Cancel J D. ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis. Plant J, 2004, 41: 353–363



[32]Larsen P B, Cancel J, Rounds M, Ochoa V. Arabidopsis ALS1 encodes a root tip and stele localized half type ABC transporter required for root growth in an aluminum toxic environment. Planta, 2007, 225: 1447–1458



[33]Hamm H E. The many faces of G protein signaling. J Biol Chem, 1998, 273: 669–672



[34]Adman E T. Structure and function of copper-containing proteins. Curr Opin Struct Biol, 1991, 1: 895–904



[35]Joo J H, Wang S, Chen J, Jones A, Fedoroff N V. Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone. Plant Cell Onl, 2005, 17: 957–970



[36]Delhaize E, Ryan P R, Hebb D M, Yamamoto Y, Sasaki T, Matsumoto H. Engineering high-level aluminum tolerance in barley with the ALMT1 gene. Proc Natl Acad Sci USA, 2004, 101: 15249–15254



[37]Larsen P B, Degenhardt J, Tai C Y, Stenzler L M, Howell S H, Kochian L V. Aluminum-resistant Arabidopsis mutants that exhibit altered patterns of aluminum accumulation and organic acid release from roots. Plant Physiol, 1998, 117: 9–17



[38]Iuchi S, Koyama H, Iuchi A, Kobayashi Y, Kitabayashi S, Kobayashi Y, Ikka T, Hirayama T, Shinozaki K, Kobayashi M. Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. Proc Natl Acad Sci USA, 2007, 104: 9900–9905



[39]Xia J, Yamaji N, Kasai T, Ma J F. Plasma membrane-localized transporter for aluminum in rice. Proc Natl Acad Sci USA, 2010, 107: 18381–18385

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