Overexpression of Zoysia ZjCIGR1 gene confers cold stress resistance to zoysiagrass

  • Yang-Ji Kim
  • Dae-Hwa YangEmail author
  • Mi-Young Park
  • Hyeon-Jin Sun
  • Pill-Soon Song
  • Hong-Gyu Kang
  • Seok-Cheol Suh
  • Yong-Eok Lee
  • Hyo-Yeon LeeEmail author
Original Article


Zoysia japonica Steud. is a warm-season lawn grass popular in Korea and elsewhere. They are cultivated in many places such as river banks, roadside, and play grounds. However, there still is a disadvantage of frequent mowing, and the grass grows poorly under the chilly conditions. To develop a grass variety that circumvents these drawbacks, we cloned the chitin-inducible gibberellins-responsive1 gene (CIGR1) from zoysiagrass. The full length of the ZjCIGR1 (Zj; Zoysia japonica Steud.) gene was obtained by 5′/3′ RACE PCR and the phylogenetic tree showed that it belonged to the CIGR1-subgroup in the PAT1-group of GRAS protein family. Expression of the ZjCIGR1 in wild-type plants was confirmed in roots, meristems, leaves, and flowers, especially high in the flowers. The transgenic zoysiagrass was confirmed by PCR using gene-specific primers, phosphinothricin-acetyl-transferase (PAT) strip test, and Southern blot analysis. ZjCIGR1-overexpressing plants acquired tolerance to cold stress displaying morphological phenotypes characteristic of stress resistance. In addition, in the transformants, expression of the ZjCIGR1 as well as cold-regulated (COR) gene was increased compared to the wild-type plants under cold stress condition. These results suggest that ZjCIGR1 gene is an important candidate for regulating cold stress resistance.


ZjCIGR1 Cold stress Transgenic zoysiagrass GRAS protein family 



Phosphinothricin acetyltransferase




Reverse transcriptase polymerase chain reaction


Transgenic third generation


Zoysia japonica chitin-inducible gibberellin-responsive 1 gene


CaMV 35S promoter



This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A6A1A03012862), and by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bioindustry Technology Development Project, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA; Grant Number: 315025-3). The grantors had not played any role in writing this report or the decision to submit this article for publication.

Supplementary material

11816_2019_570_MOESM1_ESM.doc (498 kb)
Supplementary material 1 (DOC 497 kb)


  1. Bae EJ, See KS, Kim DS, Han EH, Lee SM, Lee DW (2013) Sod production and current status of cultivation management in Korea. Weed Turf Sci 2:95–99CrossRefGoogle Scholar
  2. Bae EJ, Han JJ, Lee KS, Park YB, Chi SM (2016) Growth characteristic of warm-season turfgrass in Saemangeum reclaimed land. KSOERT 19:13–23Google Scholar
  3. Bolle C (2004) The role of GRAS proteins in plant signal transduction and development. Planta 218:683–692PubMedCrossRefPubMedCentralGoogle Scholar
  4. Bolle C, Koncz C, Chua NH (2000) PAT1, a new member of the GRAS family, is involved in phytochrome A signal transduction. Genes Dev 14:1269–1278PubMedPubMedCentralGoogle Scholar
  5. Cabassa-Hourton C, Schertl P, Bordenave-Jacquemin B, Saadallah K, Guivarc’h A, Lebreton S, Planchais S, Klodmann J, Eubel H, Crilat E, Lefebvre-De Vos D, Ghelis T, Richard L, Abdelly C, Carol P, Braun HP, Savoure A (2016) Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana. Biochem J 473:2623–2634PubMedCrossRefPubMedCentralGoogle Scholar
  6. Chai B, Maqbool SB, Hajela RK, Green D, Vargas JM Jr, Warkentin D, Sabzikar R, Sticklen MB (2002) Cloning of a chitinase-like cDNA (hs2), its transfer to creeping bentgrass (Agrostis palustris Huds.) and development of brown patch (Rhizoctonia solani) disease-resistant transgenic lines. Plant Sci 163:183–193CrossRefGoogle Scholar
  7. Cho KC, Han YJ, Kim SJ, Lee SS, Hwang OJ, Song PS, Kim JI (2011) Resistance to Rhizoctonia solani AG-2-2 (IIIB) in creeping bentgrass plants transformed with pepper esterase gene PepEST. Plant Pathol 1:1–9Google Scholar
  8. Chung SJ, Choi YI, Lee GJ (2013) Miscanthus EST-originated transcription factor WRKY expression in response to low temperature in warm-season turfgrasses. Weed Turf Sci 2:368–375CrossRefGoogle Scholar
  9. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefGoogle Scholar
  10. Czikkel BE, Maxwell DP (2007) NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes to the nucleus. Plant Physiol 164:1220–1230CrossRefGoogle Scholar
  11. Day RB, Shibuya N, Minami E (2003) Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor. Biochim Biophys Acta 1625:261–268PubMedCrossRefPubMedCentralGoogle Scholar
  12. Day RB, Tanabe S, Koshioka M, Mitsui T, Itoh H, Ueguchi-Tanaka U, Matsuoka M, Kaku H, Shibuya N, Minami E (2004) Two rice GRAS family genes responsive to N-acetylchitooligosaccharide elicitor are induced by phytoactive gibberellins: evidence for cross-talk between elicitor and gibberellins signaling in rice cells. Plant Mol Biol 54:261–272PubMedCrossRefPubMedCentralGoogle Scholar
  13. Fode B, Siemsen T, Thurow C, Weigel R, Gatz C (2008) The Arabidopsis GRAS protein SCL14 interacts with class II TGA transcription factors and is essential for the activation of stress-inducible promoters. Plant Cell 20:3122–3135PubMedPubMedCentralCrossRefGoogle Scholar
  14. Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14:1675–1690PubMedPubMedCentralCrossRefGoogle Scholar
  15. Fu D, Tisserat NA, Xiao Y, Settle D, Muthukrishnan S, Liang GH (2005) Overexpression of rice TLPD34 enhances dollar-spot resistance in transgenic bentgrass. Plant Sci 168:671–680CrossRefGoogle Scholar
  16. Fu D, Huang B, Xiao Y, Muthukrishnan S, Liang GH (2007) Overexpression of barley hva1 gene in creeping bentgrass for improving drought resistance. Plant Cell Rep 26:467–477PubMedCrossRefPubMedCentralGoogle Scholar
  17. Ganeshan S, Vitamvas P, Flowler B, Chibbar RN (2008) Quantitative expression analysis of selected COR genes reveals their differential expression in leaf and crown tissues of wheat (Triticum aestivum L.) during an extended low temperature acclimation regimen. J Exp Bot 59:2393–2402PubMedPubMedCentralCrossRefGoogle Scholar
  18. Gao MJ, Parkin IAP, Lydiate DJ, Hannoufa A (2004) An auxin-responsive SCARECROW-like transcriptional activator interacts with histone deacetylase. Plant Mol Biol 55:417–431PubMedCrossRefPubMedCentralGoogle Scholar
  19. Greb T, Clarenz O, Schafer E, Muller D, Herrero R, Schmitz G, Theres K (2003) Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev 17:1175–118711PubMedPubMedCentralCrossRefGoogle Scholar
  20. Guo Z, Bonos S, Meyer WA, Day PR, Belanger FC (2003) Transgenic creeping bentgrass with delayed dollar-spot symptoms. Mol Breed 11:95–101CrossRefGoogle Scholar
  21. Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235CrossRefGoogle Scholar
  22. Han YJ, Kim YM, Lee JY, Kim SJ, Cho KC, Chandrasekhar T, Song PS, Woo YM, Kim JI (2009) Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation. Plant Cell Rep 28:397–406PubMedCrossRefGoogle Scholar
  23. Hao Y, Cui H (2012) SHORT-ROOT regulates vascular patterning, but not apical meristematic activity in the Arabidopsis root through cytokinin homeostasis. Plant Signal Behav 7:1–4CrossRefGoogle Scholar
  24. Heery DM, Kalkhoven E, Hoare S, Parker MG (1997) A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736PubMedCrossRefGoogle Scholar
  25. Hou X, Yen L, Lee C, Xia K, Yan Y, Yu H (2010) DELLAs modulate jasmonate signalling via competitive binding to JAZs. Dev Cell 19:884–894PubMedCrossRefGoogle Scholar
  26. Hung CJ, Ginzinger DG, Zamegar R, Kanauchi H, Wong MG, Kebeew E, Clark OH, Duh QY (2003) Expression of vascular endothelial growth factor-C in benign and malignant thyroid tumors. J Clin Endocrinol Metabol 88:3694–3699CrossRefGoogle Scholar
  27. Itoh H, Ueguchi-Tanaka M, Sato Y, Ashikari M, Matsuoka M (2002) The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei. Plant Cell 14:57–70PubMedPubMedCentralCrossRefGoogle Scholar
  28. Kalo P, Gleason C, Edward A, Marsh J, Mitra RM, Hirsch S, Jakab J, Sims S, Long SR, Rogers J, Kiss GB, Downie JA, Oldroyd GE (2005) Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308:1786–1789PubMedCrossRefPubMedCentralGoogle Scholar
  29. Kim SJ, Lee JY, Kim YM, Yang SS, Hwang OJ, Hong NJ, Kim KM, Lee HY (2007) Agrobacterium-mediated high-efficiency transformation of creeping bentgrass with herbicide resistance. J Plant Biol 50:577–585CrossRefGoogle Scholar
  30. Koczula KM, Gallotta A (2016) Lateral flow assays. Essays Biochem 60:111–120PubMedPubMedCentralCrossRefGoogle Scholar
  31. Koizumi K, Hayashi T, Wu S, Gallagher KL (2012) The SHORT-ROOT protein acts as a mobile, dose-dependent signal in pattering the ground tissue. Proc Natl Acad Sci USA 109:13010–13015PubMedCrossRefPubMedCentralGoogle Scholar
  32. Kovi MR, Zhang Y, Yu S, Yang G, Yan W, Xing Y (2011) Candidacy of a chitin-inducible gibberellins-responsive gene for a major locus affecting plant height in rice that is closely linked to Green Revolution gene sd1. Theor Appl Genet 123:705–714PubMedCrossRefPubMedCentralGoogle Scholar
  33. Lata C, Prasad M (2011) Role of DREBs in regulation of abiotic stress responses in plants. J Exp Bot 62:4731–4748PubMedCrossRefPubMedCentralGoogle Scholar
  34. Lee LY, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiol 146:325–332PubMedPubMedCentralCrossRefGoogle Scholar
  35. Lee MH, Kim B, Song SK, Heo JO, Yu NI, Lee SA, Kim M, Kim DG, Sohn SO, Lim CE, Chang KS, Lee MM, Lim J (2008) Large-scale analysis of the GRAS gene family in Arabidopsis thaliana. Plant Mol Biol 67:659–670PubMedCrossRefPubMedCentralGoogle Scholar
  36. Lee SY, Boon NJ, Webb AAR, Tanaka RJ (2016) Synergistic Activation of RD29A via integration of salinity stress and abscisic acid in Arabidopsis thaliana. Plant Cell Physiol 57:2147–2160PubMedPubMedCentralCrossRefGoogle Scholar
  37. Lim PO, Kim Y, Breeze E, Koo JC, Woo HR, Ryu JS, Beynon J, Tabrett A, Buchanan-Wollaston V, Nam HG (2007) Overexpression of a chromatin architecture-controlling AT-hook protein extends leaf longevity and increases the post-harvest storage life of plants. Plant J 52:1140–1153PubMedCrossRefPubMedCentralGoogle Scholar
  38. Lin C, Thomanshow M (1992) DNA sequence analysis of a complementary DNA for cold-regulated Arabidopsis gene cor15 and characterization of the COR15 polypeptide. Plant Physiol 99:519–525PubMedPubMedCentralCrossRefGoogle Scholar
  39. Lucas MD, Daviere JM, Falcon MR, Pontin M, Iglesias-Pedraz JM, Lorrain S, Fankhauser C, Blazques MA, Titarenko E, Prat S (2008) A molecular framework for light and gibberellins control of cell elongation. Nature 451:480–486PubMedCrossRefPubMedCentralGoogle Scholar
  40. Ma HS, Liang D, Shuai P, Xia XL, Yin WL (2010) The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana. J Exp Bot 61:4011–4019PubMedPubMedCentralCrossRefGoogle Scholar
  41. Maruyama K, Sakuma Y, Kasuga M, Ito Y, Seki M, Goda H, Shimada Y, Yoshida S, Shinozaki K, Yamaquchi-Shinozaki K (2004) Identification of cold-inducible down-stream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J 38:982–993PubMedCrossRefPubMedCentralGoogle Scholar
  42. Miura K, Ohta M, Nakazawa M, Ono M, Hasegawa PM (2011) ICE Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance. Plant J 67:269–279PubMedCrossRefPubMedCentralGoogle Scholar
  43. Msanne J, Lin J, Stone JM, Awada T (2011) Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta 234:97–107PubMedCrossRefPubMedCentralGoogle Scholar
  44. Murase K, Hirano Y, Sun TP, Hakoshima T (2008) Gibberellin-induced DELLA recognition by the gibberellins receptor GID1. Nature 456:459–464PubMedCrossRefPubMedCentralGoogle Scholar
  45. Nakayama K, Okawa K, Kakizaki T, Honma T, Itoh H, Inaba T (2007) Arabidopsis Cor15am is a chloroplast stromal protein that has cryoprotective activity and forms oligomers. Plant Physiol 144:513–523PubMedPubMedCentralCrossRefGoogle Scholar
  46. Novillo F, Medina J, Salinas J (2007) Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon. Proc Natl Acad Sci USA 104:21002–21007PubMedCrossRefPubMedCentralGoogle Scholar
  47. Ogawa M, Kusano T, Katsumi M, Sano H (2000) Rice gibberellins-insensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level. Gene 245:21–29PubMedCrossRefPubMedCentralGoogle Scholar
  48. Peng J, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP, Harberd NP (1997) The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellins responses. Genes Dev 11:3194–3205PubMedPubMedCentralCrossRefGoogle Scholar
  49. Plett D, Safwat G, Gilliham M, Moller IS, Roy S, Shirley N, Jacobs A, Johnson A, Tester M (2010) Improved salinity tolerance of rice through cell type-specific expression of AtHKT1;1. PLoS ONE 5:e12571PubMedPubMedCentralCrossRefGoogle Scholar
  50. Pysh LD, Wysocka-Diller JW, Camilleri C, Bouchez D, Benfey PN (1999) The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J 18:111–119PubMedCrossRefPubMedCentralGoogle Scholar
  51. Qamar A, Mysore KS, Senthil-Kumar M (2015) Role of proline and pyrroline-5-carboxylate metabolism in plant defense against invading pathogens. Front Plant Sci 6:503–511PubMedPubMedCentralCrossRefGoogle Scholar
  52. Sanchez C, Vielba JM, Ferro E, Covelo G, Sole A, Abarca D, Mier BS, Diaz-Sala C (2007) Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species. Tree Physiol 27:1459–1470PubMedCrossRefPubMedCentralGoogle Scholar
  53. Schumacher K, Schmitt T, Rossberg M, Schmitz G, Theres K (1998) The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family. Plant Biol 96:290–295Google Scholar
  54. Silverstone AL, Ciampaglio CN, Sun T (1998) The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell 10:155–169PubMedPubMedCentralCrossRefGoogle Scholar
  55. Song IJ, Sun HJ, Jeong OK, Yang DH, Jin ID, Kang HG, Ko SM, Kwon YI, Bae TW, Song PS, Lee HY (2017) Development of dwarf type cultivar ‘Halla Green 2’ in Zoysia japonica Steud. KSOBS 49:31–35Google Scholar
  56. Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA 95:14570–14575PubMedCrossRefPubMedCentralGoogle Scholar
  57. Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040PubMedCrossRefPubMedCentralGoogle Scholar
  58. Sun X, Jones WT, Rikkerink HA (2012) GRAS proteins: the versatile roles of intrinsically disordered proteins in plant signaling. Biochemistry 442:1–12CrossRefGoogle Scholar
  59. Tester M, Bacic A (2005) Abiotic stress tolerance in Grasses from model plants to crop plants. Plant Physiol 137:791–793PubMedPubMedCentralCrossRefGoogle Scholar
  60. Thalhammer A, Bryant G, Sulpice R, Hincha DK (2014) Disordered cold regulated 15 proteins protect chloroplast membranes during freezing through binding and folding, but do not stabilize chloroplast enzymes in vivo. Plant Physiol 166:190–201PubMedPubMedCentralCrossRefGoogle Scholar
  61. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 25:4876–4882PubMedCrossRefGoogle Scholar
  62. Tian G, Wan P, Sun S, Li J, Chen M (2004) Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Mol Biol 54:519–532PubMedCrossRefGoogle Scholar
  63. Torres-Galea P, Hirtreiter B, Bolle C (2013) Two GRAS proteins, SCARECROW-LIKE21 and PHYTOCHROME A SIGNAL TRANSDUCTION1, function cooperatively in phytochrome A signal transduction. Plant Physiol 161:291–304PubMedCrossRefGoogle Scholar
  64. Toyama K, Bae CB, Kang JG, Lim YP, Adachi T, Riu KZ, Song PS, Lee HY (2003) Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation. Mol Cell 16:19–27Google Scholar
  65. Wang Y, Kausch AP, Chandlee JM, Luo H, Ruemmele BA, Browning M, Jackson N, Goldsmith MR (2003) Co-transfer and expression of chitinase, glucanase, and bar genes in creeping bentgrass for conferring fungal disease resistance. Plant Sci 165:497–506CrossRefGoogle Scholar
  66. Wang J, Liu S, Li C, Wang T, Zhang P, Chen K (2017) PnLRR-RLK27, a novel leucine-rich repeats receptor-like protein kinase from the Antarctic moss Pohlia nutans, positively regulates salinity and oxidation-stress tolerance. PLoS ONE 12:e0172869PubMedPubMedCentralCrossRefGoogle Scholar
  67. Wei SJ, Du ZL, Gao F, Ke X, Li J, Liu JX, Zhou YJ (2015) Global transcriptome profiles of ‘Meyer’ zoysiagrass in response to cold stress. PLoS ONE 10(6):e0130053CrossRefGoogle Scholar
  68. Woo HR, Kim JH, Nam HG, Lim PO (2004) The delayed leaf senescence mutants of Arabidopsis, ore1, ore3, and ore9 are tolerant to oxidative stress. Plant Cell Physiol 45:923–932PubMedCrossRefGoogle Scholar
  69. Xiang DJ, Hu XY, Zhang Y, Yin KD (2008) Over-expression of ICE1 gene in transgenic rice improves cold tolerance. Rice Sci 15:173–178CrossRefGoogle Scholar
  70. Xu K, Chen S, Li T, Ma X, Liang X, Ding X, Liu H, Luo L (2015) OsGRAS23, a rice GRAS transcription factor gene, is involved in drought stress response through regulating expression of stress-responsive genes. BMC Plant Biol 15:141–153PubMedPubMedCentralCrossRefGoogle Scholar
  71. Yang DH, Sun HJ, Goh CH, Song PS, Bae TW, Song IJ, Lim YP, Lim PO, Lee HY (2011) Cloning of Zoysia ZjLsL and its overexpression to induce axillary meristem initiation and tiller formation in Arabidopsis and bentgrass. Plant Biol 14:411–419PubMedCrossRefPubMedCentralGoogle Scholar
  72. Yu TT, Skinner DZ, Liang GH, Trick HN, Huang B, Muthukrishnan S (2000) Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas 133:229–233PubMedCrossRefPubMedCentralGoogle Scholar
  73. Yuan Y, Fang L, Karungo SK, Zhang L, Gao Y, Li S, Xin H (2015) Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis. Plant Cell Rep 35:655–666PubMedCrossRefPubMedCentralGoogle Scholar
  74. Zhong H, Boyland MG, Srinivasan C, Sticklen MB (1993) Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Rep 13:1–6 PubMedCrossRefPubMedCentralGoogle Scholar
  75. Zhuang L, Yuan X, Chen Y, Xu B, Yang Z, Huang B (2015) PpCBF3 from cold-tolerant kentucky bluegrass involved in freezing tolerance associated with up-regulation of cold-related genes in transgenic Arabidopsis thaliana. PLoS ONE 10(7):e0132928PubMedPubMedCentralCrossRefGoogle Scholar

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© Korean Society for Plant Biotechnology 2019

Authors and Affiliations

  1. 1.Faculty of BiotechnologyJeju National UniversityJejuRepublic of Korea
  2. 2.Subtropical Horticulture Research InstituteJeju National UniversityJejuRepublic of Korea
  3. 3.Subtropical/Tropical Organism Gene BankJeju National UniversityJejuRepublic of Korea
  4. 4.Department of BiotechnologyDongguk UniversityGyeongjuRepublic of Korea

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