Abstract
Arabidopsis GIGANTEA (GI) is encoded by a single gene and highly conserved among vascular plants and its mutants display pleiotropic phenotypes involved in diverse biological processes such as light signaling, circadian clock, and sucrose metabolism as well as abiotic stress responses. However, molecular mechanisms of GI are largely unknown due to the lack of useful antibody. To date, the epitope tags have been widely used to detect GI in plants, but it needs to generate the transgenic plants which take a few months. Here, we produced polyclonal α-GI antibody using truncated variants of GI having amino-terminal (1–858 aa) and carboxyl-terminal (920–1173) regions as antigens. Both recombinant His-GI1-858 and His-GI920–1173 proteins were individually and successfully expressed in E. coli and immunized into rabbit. Anti-serum was purified by antigenspecific affinity purification method using both recombinant His-GI1–858 and His-GI920–1173 proteins. Purified polyclonal α-GI antibody not only detected endogenous GI proteins in wild-type Arabidopsis plants, but also reenacted its diel oscillations. Furthermore, the antibody showed cross-reactivity with the GI orthologs in other plants such as Chinese cabbage, rape and tomato. Our polyclonal GI antibody could help to determine the molecular mechanisms of GI involved in largely unknown pleiotropic responses in plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Black MM, Stockum C, Dickson JM, Putterill J, Arcus VL (2011) Expression, purification and characterization of GIGANTEA: a circadian clock-controlled regulator of photoperiodic flowering in plants. Protein Expr Purif 76:197–204
Bendix C, Mendoza JM, Stanley DN, Meeley R, Harmon FG (2013) The circadian clock-associated gene gigantea1 affects maize developmental transitions. Plant Cell Environ 34:1379–1390
Cha JY, Kim J, Kim TS, Zeng Q, Wang L, Lee SY, Kim WY, Somers DE (2017) GIGANTEA is a co-chaperone which facilitates maturation of ZEITLUPE in the Arabidopsis circadian clock. Nature Commun 8:3
Corellou F, Schwartz C, Motta JP, Djouani-Tahri el B, Sanchez F, Bouget FY (2009) Clocks in the green lineage: Comparative functional analysis of the circadian architecture of the picoeukaryote Ostreococcus. Plant Cell 21:3436–3449
David KM, Armbruster U, Tama N, Putterill J (2006) Arabidopsis GIGANTEA protein is post-transcriptionally regulated by light and dark. FEBS Lett 580:1193–1197
Dunford RP, Griffiths S, Christodoulou V, Laurie DA (2005) Characterization of a barley (Hordeum vulgare L.) homologue of the Arabidopsis flowering time regulator GIGANTEA. Theor Appl Genet 110:925–931
Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B, Coupland G, Putterill J (1999) GIGANTEA: a circadian clockcontrolled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membranespanning domains. EMBO J 18:4679–4688
Hayama R, Izawa T, Shimamoto K (2002) Isolation of rice genes possibly involved in the photoperiodic control of flowering by a fluorescent differential display method. Plant Cell Physiol 43:494–504
Hecht V, Knowles CL, Vander Schoor JK, Liew LC, Jones SE, Lambert MJM, Weller JL (2007) Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs. Plant Physiol 144:648–661
Higuchi Y, Sage-Ono K, Sasaki R, Ohtsuki N, Hoshino A, Iida S, Kamada H, Ono M (2011) Constitutive expression of the GIGANTEA ortholog affects circadian rhythms and suppresses one-shot induction of flowering in Pharbitis nil, a typical shortday plant. Plant Cell Physiol 52:638–650
Hong SY, Lee S, Seo PJ, Yang MS, Park CM (2010) Identification and molecular characterization of a Brachypodium distachyon GIGANTEA gene: functional conservation in monocot and dicot plants. Plant Mol Biol 72:485–497
Ke Q, Kim HS, Wang Z, Ji CY, Jeong JC, Lee HS, Choi YI, Xu B, Deng X, Yun DJ, Kwak SS (2017) Down-regulation of GIGANTEAlike genes increases plant growth and salt stress tolerance in poplar. Plant Biotechnol J 15:331–343
Kim JA, Jung H, Hong JK, Hermand V, McClung CR, Lee YH, Kim JY, Lee SI, Jeong MJ, Kim J, Yum DJ, Kim WY (2016) Reduction of GIGANTEA expression in transgenic Brassica rapa enhances salt tolerance. Plant Cell Rep 35:1943–1954
Kim WY, Ali Z, Park HJ, Park SJ, Cha JY, Perez-Hormaeche J, Quintero FJ, Shin G, Kim MR, Qiang Z, Ning L, Park HC, Lee SY, Bressan RA, Pardo JM, Bohnert HJ, Yun DJ (2013) Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in Arabidopsis. Nature Commun 4:1352
Kim WY, Fujiwara S, Suh SS, Kim J, Kim Y, Han L, David K, Putterill J, Nam HG, Somers DE (2007) ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light. Nature 449:356–360
Kubota A, Kita S, Ishizaki K, Nishihama R, Yamato KT, Kohchi T (2014) Co-option of a photoperiodic growth-phase transition system during land plant evolution. Nature Commun 5:3668
Li F, Zhang X, Hu R, Wu F, Ma J, Meng Y, Fu YF (2013) Identification and molecular characterization of FKF1 and GI homologous genes in soybean. PLoS ONE 8:e79036
Linde AM, Eklund DM, Kubota A, Pederson ERA, Holm K, Gyllenstrand N, Nishihama R, Cronberg N, Muranaka T, Oyama T, Kohchi T, Lagercrantz U (2017) Early evolution of the land plant circadian clock. New Phytol doi: 10.1111/nph.14487
López-Torrejón G, Guerra D, Catalá R, Salinas J, del Pozo JC (2013) Identification of SUMO targets by a novel proteomic approach in plants. J Integr Plant Biol 55:96–107
Martin-Tryon EL, Kreps JA, Harmer SL (2007) GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation. Plant Physiol 143:473–486
Mas P, Kim WY, Somers DE, Kay SA (2003) Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426:567–570
Mishra P, Panigrahi KC (2015) GIGANTEA-an emerging story. Front Plant Sci 6:8
Paltiel J, Amin R, Gover A, Ori N, Smach A (2006) Novel roles for GIGANTEA revealed under environmental conditions that modify its expression in Arabidopsis and Medicago truncatula. Planta 224:1255–1268
Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, Kim HJ, Kay SA, Nam HG (1999) Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science 285:1579–1582
Somers DE, Schultz TF, Milnamow M, Kay SA (2000) ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell 101:319–329
Tang W, Yan H, Su ZX, Park SC, Liu YJ, Zhang YG, Wang X, Kou M, Ma DF, Kwan SS, Li Q (2017) Cloning and characterization of a novel GIGANTEA gene in sweet potato. Plant Physiol Biochem 116:27–35
Xie Q, Lou P, Hermand V, Aman R, Park HJ, Yun DJ, Kim WY, Salmela MJ, Ewers BE, Weinig C, Khan SL, Schaible DLP, McClung CR (2015) Allelic polymorphism of GIGANTEA is responsible for naturally occurring variation in circadian period in Brassica rapa. Proc Natl Acad Sci USA 112:3829–3834
Zhao XY, Liu MS, Li JR, Guan CM, Zhang XS (2005) The wheat TaGI1, involved in photoperiodic flowering, encodes an Arabidopsis GI ortholog. Plant Mol Biol 58:53–64
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Khaleda, L., Cha, JY., Kim, M.G. et al. Production and characterization of polyclonal antibody against Arabidopsis GIGANTEA, a circadian clock controlled flowering time regulator. J. Plant Biol. 60, 622–629 (2017). https://doi.org/10.1007/s12374-017-0305-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12374-017-0305-7