Plant Cell Reports

, Volume 38, Issue 9, pp 1109–1125 | Cite as

Over-expression of the Brachypodium ASR gene, BdASR4, enhances drought tolerance in Brachypodium distachyon

  • Jin Seok Yoon
  • Jae Yoon Kim
  • Man Bo Lee
  • Yong Weon SeoEmail author
Original Article


Key message

BdASR4 expression was up-regulated during abiotic stress and hormone treatments. Plants over-expressing BdASR4 improved drought tolerant. BdASR4 may regulate antioxidant activities and transcript levels of stress-related and abscisic acid-responsive genes.


Abiotic stress conditions negatively affect plant growth and developmental processes, causing a reduction in crop productivity. The abscisic acid-, stress-, ripening-induced (ASR) proteins play important roles in the protection of plants from abiotic stress. Brachypodium distachyon L. is a well-studied monocot model plant. However, ASR proteins of Brachypodium have not been widely studied. In this study, five ASR genes of Brachypodium plant were cloned and characterized. The BdASR genes were expressed in response to various abiotic stresses and hormones. In particular, BdASR4 was shown to encode a protein containing a nuclear localization signal in its C-terminal region, which enabled protein localization in the nucleus. To further examine functions of BdASR4, transgenic Brachypodium plants harboring BdASR4 were generated. Over-expression of BdASR4 was associated with strong drought tolerance, and plants over-expressing BdASR4 preserved more water and displayed higher antioxidant enzyme activities than did the wild-type plants. The transcript levels of stress-responsive genes, reactive oxygen species scavenger-associated genes, and abscisic acid-responsive genes tended to be higher in transgenic plants than in WT plants. Moreover, plants over-expressing BdASR4 were hypersensitive to exogenous abscisic acid at the germination stage. Taken together, these findings suggest multiple roles for BdASR4 in the plant response to drought stress by regulating antioxidant enzymes and the transcription of stress- and abscisic acid-responsive genes.


Drought Brachypodium distachyon L. Abscisic acid Enzymatic antioxidants Stress-responsive genes 



Abscisic acid


Aba-, stress-, ripening-induced


Ascorbate peroxidase


Green fluorescent protein




Polyethylene glycol


Relative water content


Superoxide dismutase



This work was supported by the Rural Development Administration, Republic of Korea through their project “Cooperative Research Program for Agriculture Science & Technology Development (Project no. PJ013244)”.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2019_2429_MOESM1_ESM.docx (2.4 mb)
Supplementary material 1 (DOCX 2478 kb)


  1. Abe H, Tanka S, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alves SC, Worland B, Thole V, Snape JW, Bevan MW, Vain P (2009) A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat Protoc 4:638CrossRefPubMedGoogle Scholar
  3. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399CrossRefPubMedGoogle Scholar
  4. Arenhart RA, De Lima JC, Pedron M, Carvalho FE, Silveira JA, Rosa SB, Caverzan A, Andrade CM, Schunemann M, Margis R, Margis-Pinheiro M (2013) Involvement of ASR genes in aluminium tolerance mechanisms in rice. Plant Cell Environ 36:52–67CrossRefPubMedGoogle Scholar
  5. Arenhart RA, Bai Y, Valter de Oliveria LF, Bucker Neto L, Schunemann M, Maraschin FS, Mariath J, Silverio A, Sachetto-Martins G, Margis R, Wang ZY, Margis-Pinheiro M (2014) New insights into aluminum tolerance in rice: the ASR5 protein binds the STAR1 promoter and other aluminum-responsive genes. Mol Plant 7:709–721CrossRefPubMedGoogle Scholar
  6. Barrs H, Weatherley P (1962) A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust J Biol Sci 15:413–428CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedPubMedCentralGoogle Scholar
  8. Çakir B, Agasse A, Gaillard C, Saumonneau A, Delrot S, Atanassova R (2003) A grape ASR protein involved in sugar and abscisic acid signaling. Plant Cell 15:2165–2180CrossRefPubMedPubMedCentralGoogle Scholar
  9. Carrari F, Fernie AR, Iusem ND (2004) Heard it through the grapevine? ABA and sugar cross-talk: the ASR story. Trend Plant Sci 9:57–59CrossRefGoogle Scholar
  10. Chen H, Nelson R, Sherwood J (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16(664–668):670Google Scholar
  11. Chen J-Y, Liu D-J, Jiang Y-M, Zhao M-L, Shan W, Kuang J-F, Lu W-J (2011) Molecular characterization of a strawberry FaASR gene in relation to fruit ripening. PLoS One 6:e24649CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9:436–442CrossRefPubMedGoogle Scholar
  13. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930CrossRefPubMedGoogle Scholar
  14. Golan I, Dominguez PG, Konrad Z, Shkolnik-Inbar D, Carrari F, Bar-Zvi D (2014) Tomato abscisic acid stress ripening (ASR) gene family revisited. PLoS One 9:e107117CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hajela RK, Horvath DP, Gilmour SJ, Thomashow MF (1990) Molecular cloning and expression of cor (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol 93:1246–1252CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hong MJ, Kim DY, Seo YW (2013) SKP1-like-related genes interact with various F-box proteins and may form SCF complexes with Cullin–F-box proteins in wheat. Mol Biol Rep 40:969–981CrossRefPubMedGoogle Scholar
  17. Hu W, Huang C, Deng X, Zhou S, Chen L, Li Y, Wang C, Ma Z, Yuan Q, Wnag Y, Cai R, Liang X, Yang G, He G (2013) TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environ 36:1449–1464CrossRefPubMedGoogle Scholar
  18. Iusem ND, Bartholomew DM, Hitz WD, Scolnik PA (1993) Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiol 102:1353CrossRefPubMedPubMedCentralGoogle Scholar
  19. Jeffreys A, Flavell R (1977) A physical map of the DNA regions flanking the rabbit β-globin gene. Cell 12:429–439CrossRefPubMedGoogle Scholar
  20. Jha B, Lal S, Tiwari V, Yadav SK, Agarwal PK (2012) The SbASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco. Mar Biotechnol (NY) 14:782–792CrossRefGoogle Scholar
  21. Joo JH, Wang S, Chen J, Jones A, Fedoroff NV (2005) Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone. Plant Cell 17:957–970CrossRefPubMedPubMedCentralGoogle Scholar
  22. Joo J, Lee YH, Kim Y-K, Nahm BH, Song SI (2013) Abiotic stress responsive rice ASR1 and ASR3 exhibit different tissue-dependent sugar and hormone-sensitivities. Mol Cells 35:421–435CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kalifa Y, Perlson E, Gilad A, Konrad Z, Scolnik P, Bar-Zvi D (2004) Over-expression of the water and salt stress-regulated Asr1 gene confers an increased salt tolerance. Plant Cell Environ 27:1459–1468CrossRefGoogle Scholar
  24. Kang H, Zhang M, Zhou S, Guo Q, Chen F, Wu J, Wang W (2016) Overexpression of wheat ubiquitin gene, Ta-Ub2, improves abiotic stress tolerance of Brachypodium distachyon. Plant Sci 248:102–115CrossRefPubMedGoogle Scholar
  25. Kim S-J, Lee S-C, Hong SK, An K, An G, Kim S-R (2009) Ectopic expression of a cold-responsive OsAsr1 cDNA gives enhanced cold tolerance in transgenic rice plants. Mol Cells 27:449–458CrossRefPubMedGoogle Scholar
  26. Kim DY, Hong MJ, Park C-S, Seo YW (2015) The effects of chronic radiation of gamma ray on protein expression and oxidative stress in Brachypodium distachyon. Intl J Radiat Biol 91:407–419CrossRefPubMedGoogle Scholar
  27. Kim YJ, Kim JY, Yoon JS, Kim DY, Hong MJ, Seo YW (2016) Characterization of 4 TaGAST genes during spike development and seed germination and their response to exogenous phytohormones in common wheat. Mol Biol Rep 43:1435–1449CrossRefPubMedGoogle Scholar
  28. Kim JY, Nong G, Rice JD, Gallo M, Preston JF, Altpeter F (2017) In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane. Plant Mol Biol 93:465–478CrossRefPubMedGoogle Scholar
  29. Lee SC, Luan S (2012) ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Environ 35:53–60CrossRefPubMedGoogle Scholar
  30. Li J, Dong Y, Li C, Pan Y, Yu J (2016a) SiASR4, the target gene of SiARDP from Setaria italica, improves abiotic stress adaption in plants. Front Plant Sci 7:2053PubMedGoogle Scholar
  31. Li J, Li Y, Yin Z, Jiang J, Zhang M, Guo X, Ye Z, Zhao Y, Xiong H, Zhang Z, Shao Y, Jiang C, Zhang H, An G, Paek NC, Ali J, Li Z (2016b) OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice. Plant Biotechnol J 15:183–196CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lim SD, Lee C, Jang CS (2014) The rice RING E3 ligase, OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis. Plant Cell Environ 37:1097–1113CrossRefPubMedGoogle Scholar
  33. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10:1391–1406CrossRefPubMedPubMedCentralGoogle Scholar
  34. Liu HY, Dai JR, Feng DR, Liu B, Wang HB, Wang JF (2010) Characterization of a novel plantain Asr gene, MpAsr, that is regulated in response to infection of Fusarium oxysporum f. sp. cubense and abiotic stresses. J Integr Plant Biol 52:315–323CrossRefPubMedGoogle Scholar
  35. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefPubMedPubMedCentralGoogle Scholar
  36. Luo L (2010) Breeding for water-saving and drought-resistance rice (WDR) in China. J Exp Bot 61:3509–3517CrossRefPubMedGoogle Scholar
  37. Miao H, Wang Y, Liu J, Jia C, Hu W, Sun P, Jin Z, Xu B (2014) Molecular cloning and expression analysis of the MaASR1 gene in banana and functional characterization under salt stress. Electron J Biotechnol 17:287–295CrossRefGoogle Scholar
  38. Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149:88–95CrossRefPubMedPubMedCentralGoogle Scholar
  39. Nakashima K, Takasaki H, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta Gene Regul Mech 1819:97–103CrossRefGoogle Scholar
  40. Pérez-Díaz J, Wu T-M, Pérez-Díaz R, Ruíz-Lara S, Hong C-Y, Casaretto JA (2014) Organ-and stress-specific expression of the ASR genes in rice. Plant Cell Rep 33:61–73CrossRefPubMedGoogle Scholar
  41. Philippe R, Courtois B, McNally KL, Mournet P, El-Malki R, Le Paslier MC, Fabre D, Billot C, Brunel D, Glaszmann JC, This D (2010) Structure, allelic diversity and selection of Asr genes, candidate for drought tolerance, in Oryza sativa L. and wild relatives. Theor Appl Genet 121:769–787CrossRefPubMedGoogle Scholar
  42. Rom S, Gilad A, Kalifa Y, Konrad Z, Karpasas MM, Goldgur Y, Bar-Zvi D (2006) Mapping the DNA-and zinc-binding domains of ASR1 (abscisic acid stress ripening), an abiotic-stress regulated plant specific protein. Biochimie 88:621–628CrossRefPubMedGoogle Scholar
  43. Rossi M, Iusem ND (1994) Tomato (Lycopersicon esculentum) genomic clone homologous to a gene encoding an abscisic acid-induced protein. Plant Physiol 104:1073–1074CrossRefPubMedPubMedCentralGoogle Scholar
  44. Ryu JY, Hong S-Y, Jo S-H, Woo J-C, Lee S, Park C-M (2014) Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon. BMC Plant Biol 14:15CrossRefPubMedPubMedCentralGoogle Scholar
  45. Saumonneau A, Agasse A, Bidoyen MT, Lallemand M, Cantereau A, Medici A, Laloi M, Atanassova R (2008) Interaction of grape ASR proteins with a DREB transcription factor in the nucleus. FEBS Lett 582:3281–3287CrossRefPubMedGoogle Scholar
  46. Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417CrossRefPubMedGoogle Scholar
  47. Shkolnik D, Bar-Zvi D (2008) Tomato ASR1 abrogates the response to abscisic acid and glucose in Arabidopsis by competing with ABI4 for DNA binding. Plant Biotechnol J 6:368–378CrossRefPubMedGoogle Scholar
  48. Thilmony R, Guttman ME, Lin JW, Blechl AE (2014) The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon. GM Crops Food 5:36–43CrossRefPubMedGoogle Scholar
  49. Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498CrossRefPubMedPubMedCentralGoogle Scholar
  50. Van Rijs J, Giguère V, Hurst J, Van Agthoven T, van Kessel AG, Goyert S, Grosveld F (1985) Chromosomal localization of the human Thy-1 gene. Proc Natl Acad Sci USA 82:5832–5835CrossRefPubMedGoogle Scholar
  51. Virlouvet L, Jacquemot MP, Gerentes D, Corti H, Bouton S, Gilard F, Valot B, Trouverie J, Tcherkez G, Falque M, Damerval C, Rogowsky P, Perez P, Noctor G, Zivy M, Coursol S (2011) The ZmASR1 protein influences branched-chain amino acid biosynthesis and maintains kernel yield in maize under water-limited conditions. Plant Physiol 157:917–936CrossRefPubMedPubMedCentralGoogle Scholar
  52. Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9:244–252CrossRefPubMedGoogle Scholar
  53. Wang L, Hu W, Feng J, Yang X, Huang Q, Xiao J, Liu Y, Yang G, He G (2016) Identification of the ASR gene family from Brachypodium distachyon and functional characterization of BdASR1 in response to drought stress. Plant Cell Rep 35:1221–1234CrossRefPubMedGoogle Scholar
  54. Warren C (2008) Rapid measurement of chlorophylls with a microplate reader. J Plant Nutr 31:1321–1332CrossRefGoogle Scholar
  55. Watt M, Schneebeli K, Dong P, Wilson IW (2009) The shoot and root growth of Brachypodium and its potential as a model for wheat and other cereal crops. Funct Plant Biol 36:960–969CrossRefGoogle Scholar
  56. Xu J, Xue C, Xue D, Zhao J, Gai J, Guo N, Xing H (2013) Overexpression of GmHsp90s, a heat shock protein 90 (Hsp90) gene family cloning from soybean, decrease damage of abiotic stresses in Arabidopsis thaliana. PLoS One 8:e69810CrossRefPubMedPubMedCentralGoogle Scholar
  57. Yamaguchi-Shinozaki K, Shinozaki K (1993) The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. Mol Gen Genet 238:17–25PubMedGoogle Scholar
  58. Yang C-Y, Chen Y-C, Jauh GY, Wang C-S (2005) A lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis. Plant Physiol 139:836–846CrossRefPubMedPubMedCentralGoogle Scholar
  59. Zale J, Jung JH, Kim JY, Pathak B, Karan R, Liu H, Chen X, Wu H, Candreva J, Zhau Z, Shanklin J, Altpeter F (2016) Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass. Plant Biotechnol J 14:661–669CrossRefPubMedGoogle Scholar
  60. Zhang L, Hu W, Wang Y, Feng R, Zhang Y, Liu J, Jia C, Miao H, Zhang J, Xu B, Jin Z (2015) The MaASR gene as a crucial component in multiple drought stress response pathways in Arabidopsis. Funct Integr Genom 15:247–260CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jin Seok Yoon
    • 1
  • Jae Yoon Kim
    • 1
    • 2
  • Man Bo Lee
    • 1
  • Yong Weon Seo
    • 1
    Email author
  1. 1.Department of Biosystems and BiotechnologyKorea UniversitySeoulRepublic of Korea
  2. 2.Department of Plant ResourcesKongju National UniversityYesanRepublic of Korea

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