Skip to main content
SpringerLink
Log in

The Brachypodium distachyon BdWRKY36 gene confers tolerance to drought stress in transgenic tobacco plants

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

The expression of BdWRKY36 was upregulated by drought treatment. BdWRKY36 -overexpressing transgenic tobacco increased drought tolerance by controlling ROS homeostasis and regulating transcription of stress related genes.

Abstract

WRKY transcription factor plays important roles in plant growth, development and stress response. However, the function of group IIe WRKYs is less known. In this study, we cloned and characterized a gene of group IIe WRKY, designated as BdWRKY36, from Brachypodium distachyon. Transient expression of BdWRKY36 in onion epidermal cell suggested its localization in the nucleus. Transactivation assay revealed that the C-terminal region, instead of full length BdWRKY36, had transcriptional activity. BdWRKY36 expression was upregulated by drought. Overexpression of BdWRKY36 in transgenic tobacco plants resulted in enhanced tolerance to drought stress. Physiological–biochemical indices analyses showed that BdWRKY36-overexpressing tobacco lines had lesser ion leakage (IL) and reactive oxygen species (ROS) accumulation, but higher contents of chlorophyll, relative water content (RWC) and activities of antioxidant enzyme than that in control plants under drought condition. Meanwhile expression levels of some ROS-scavenging and stress-responsive genes were upregulated in BdWRKY36-overexpressing tobacco lines under drought stress. These results demonstrate that BdWRKY36 functions as a positive regulator of drought stress response by controlling ROS homeostasis and regulating transcription of stress related genes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

CaMV:

Cauliflower mosaic virus

CAT:

Catalase

DAB:

3,3′-diaminobenzidine

DAPI:

4′,6-diamidino-2-phenylindole

DW:

Dry weight

FW:

Fresh weight

GAL4BD:

GAL4 DNA binding domain

GFP:

Green fluorescent protein

IL:

Ion leakage

LEA:

Late embryogenesis abundant

MS:

Murashige and skoog

NBT:

Nitroblue tetrazolium

NCED:

9-cis-epoxycarotenoid dioxygenase

OE:

Overexpression

ORF:

Open reading frame

POD:

Peroxidase

qRT-PCR:

Quantitative RT-PCR

ROS:

Reactive oxygen species

RT–PCR:

Reverse transcription–PCR

RWC:

Relative water content

SOD:

Superoxide dismutase

TW:

Turgid weight

VC:

Vector control

WT:

Wild type

References

  • Bhalla PL (2006) Genetic engineering of wheat-current challenges and opportunities. Trends Biotechnol 24:305–311

    Article  CAS  PubMed  Google Scholar 

  • Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH (2009) The WRKY6 transcription factor modulates phosphate1 expression in response to low Pi stress in Arabidopsis. Plant Cell 21:3554–3566

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen H, Lai ZB, Shi JW, Xiao Y, Chen ZX, Xu XP (2010) Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biol 10:281

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen LG, Song Y, Li SJ, Zhang LP, Zou CS, Yu DQ (2012) The role of WRKY transcription factors in plant abiotic stresses. Biochim Biophys Acta 1819:120–128

    Article  CAS  PubMed  Google Scholar 

  • Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206

    Article  CAS  PubMed  Google Scholar 

  • Foyer CH, Shigeoka S (2011) Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol 155:93–100

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hirayama T, Shinozaki K (2010) Research on plant abiotic stress responses in the post-genome era: past, present and future. Plant J 61:1041–1052

    Article  CAS  PubMed  Google Scholar 

  • Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SC, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231

    Article  CAS  Google Scholar 

  • Hu W, Huang C, Deng XM, Zhou SY, Chen LH, Li Y, Wang C, Ma ZB, Yuan QQ, Wang Y, Cai R, Liang XY, Yang GX, He GY (2013) TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environ 36:1449–1464

    Article  CAS  PubMed  Google Scholar 

  • Huang XS, Liu JH, Chen XJ (2010) Overexpression of PtrABF gene, a bZIP transcription factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance in tobacco via scavenging ROS and modulating expression of stress-responsive genes. BMC Plant Biol 10:230

    Article  PubMed Central  PubMed  Google Scholar 

  • Hundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genom 9:118

    Article  Google Scholar 

  • Ishiguro S, Nakamura K (1994) Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′upstream regions of genes coding for sporamin and β-amylase from sweet potato. Mol Gen Genet 244:563–571

    Article  CAS  PubMed  Google Scholar 

  • Jiang Y, Deyholos MK (2009) Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol 69:91–105

    Article  CAS  PubMed  Google Scholar 

  • Jiang MY, Zhang JH (2001) Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 42:1265–1273

    Article  CAS  PubMed  Google Scholar 

  • Jiang YJ, Liang G, Yu DQ (2012) Activated expression of WRKY57 confers drought tolerance in Arabidopsis. Mol Plant 5:1375–1388

    Article  CAS  PubMed  Google Scholar 

  • Johnson CS, Kolevski B, Smyth DR (2002) Transparent TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14:1359–1375

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Koo SC, Moon BC, Kim JK, Kim CY, Sung SJ, Kim MC, Cho MJ, Cheong YH (2009) OsBWMK1 mediates SA-dependent defense responses by activating the transcription factor OsWRKY33. Biochem Biophys Res Commun 387:365–370

    Article  CAS  PubMed  Google Scholar 

  • Li SJ, Fu QT, Huang WD, Yu DQ (2009) Functional analysis of an Arabidopsis transcription factor WRKY25 in heat stress. Plant Cell Rep 28:683–693

    Article  CAS  PubMed  Google Scholar 

  • Li SJ, Zhou X, Chen LG, Huang WD, Yu DQ (2010) Functional characterization of Arabidopsis thaliana WRKY39 in heat stress. Mol Cells 29:475–483

    Article  CAS  PubMed  Google Scholar 

  • Li C, Rudi H, Stockinger EJ, Cheng H, Cao M, Fox SE, Mockler TC, Westereng B, Fjellheim S, Rognli OA, Sandve SR (2012a) Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses. BMC Plant Biol 12:65

    Article  PubMed Central  PubMed  Google Scholar 

  • Li JB, Luan YS, Jin H (2012b) The tomato SlWRKY gene plays an important role in the regulation of defense responses in tobacco. Biochem Biophys Res Commun 427:671–676

    Article  CAS  PubMed  Google Scholar 

  • Li J, Besseau S, Törönen P, Sipari N, Kollist H, Holm L, Palva ET (2013) Defense-related transcription factors WRKY70 and WRKY54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis. New Phytol 200:457–472

    Article  CAS  PubMed  Google Scholar 

  • Liu QL, Zhong M, Li S, Pan YZ, Jiang BB, Jia Y, Zhang HQ (2013) Overexpression of a chrysanthemum transcription factor gene, DgWRKY3, in tobacco enhances tolerance to salt stress. Plant Physiol Biochem 69:27–33

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Luo X, Bai X, Sun XL, Zhu D, Liu BH, Ji W, Cai H, Cao L, Wu J, Hu MR, Liu X, Tang LL, Zhu YM (2013) Expression of wild soybean WRKY20 in Arabidopsis enhances drought tolerance and regulates ABA signaling. J Exp Bot 64:2155–2169

    Article  CAS  PubMed  Google Scholar 

  • Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55:853–867

    Article  CAS  PubMed  Google Scholar 

  • Miao Y, Laun TM, Smykowski A, Zentgraf U (2007) Arabidopsis MEKK1 can take a short cut: it can directly interact with senescence-related WRKY53 transcription factor on the protein level and can bind to its promoter. Plant Mol Biol 65:63–76

    Article  CAS  PubMed  Google Scholar 

  • Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33:453–467

    Article  CAS  PubMed  Google Scholar 

  • Mittler R (2006) Abiotic stress, the field environment and stress combination. Trends Plant Sci 11:15–19

    Article  CAS  PubMed  Google Scholar 

  • Niu CF, Wei W, Zhou QY, Tian AG, Hao YJ, Zhang WK, Ma B, Lin Q, Zhang ZB, Zhang JS, Chen SY (2012) Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell Environ 35:1156–1170

    Article  CAS  PubMed  Google Scholar 

  • Pan JW, Zhang MY, Kong XP, Xing X, Liu YK, Zhou Y, Liu Y, Sun LP, Li DQ (2012) ZmMPK17, a novel maize group D MAP kinase gene, is involved in multiple stress responses. Planta 235:661–676

    Article  CAS  PubMed  Google Scholar 

  • Qin YX, Tian YC, Han L, Yang XC (2013) Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana. Biochem Biophys Res Commun 441:476–481

    Article  CAS  PubMed  Google Scholar 

  • Ren X, Chen Z, Liu Y, Zhang H, Zhang M, Liu Q, Hong X, Zhu JK, Gong Z (2010) ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis. Plant J 63:417–429

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ross CA, Liu Y, Shen QJ (2007) The WRKY gene family in rice (Oryza sativa). J Integr Plant Biol 49:827–842

    Article  CAS  Google Scholar 

  • Roychoudhury A, Paul S, Basu S (2013) Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress. Plant Cell Rep 32:985–1006

    Article  CAS  PubMed  Google Scholar 

  • Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15:247–258

    Article  CAS  PubMed  Google Scholar 

  • Rushton DL, Tripathi P, Rabara RC, Lin J, Ringler P, Boken AK, Langum TJ, Smidt L, Boomsma DD, Emme NJ, Chen X, Finer JJ, Shen QJ, Rushton PJ (2012) WRKY transcription factors: key components in abscisic acid signaling. Plant Biotechnol J 10:2–11

    Article  CAS  PubMed  Google Scholar 

  • Shen HS, Liu CT, Zhang Y, Meng XP, Zhou X, Chu CC, Wang XP (2012) OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice. Plant Mol Biol 80:241–253

    Article  CAS  PubMed  Google Scholar 

  • Skirycz A, Vandenbroucke K, Clauw P, Maleux K, De Meyer B, Dhondt S, Pucci A, Gonzalez N, Hoeberichts F, Tognetti VB, Galbiati M, Tonelli C, Van Breusegem F, Vuylsteke M, Inzé D (2011) Survival and growth of Arabidopsis plants given limited water are not equal. Nat Biotechnol 29:212–214

    Article  CAS  PubMed  Google Scholar 

  • Song Y, Jing SJ, YU DQ (2009) Overexpression of the stress-induced OsWRKY08 improves osmotic stress tolerance in Arabidopsis. Chinese Sci Bull 54:4671–4678

    Article  CAS  Google Scholar 

  • Sun SJ, Guo SQ, Yang X, Bao YM, Tang HJ, Sun H, Huang J, Zhang HS (2010) Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice. J Exp Bot 61:2807–2818

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tang N, Zhang H, Li XH, Xiao JH, Xiong LZ (2012) Constitutive activation of transcription factor OsbZIP46 improves drought tolerance in rice. Plant Physiol 158:1755–1768

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tao Z, Kou YJ, Liu HB, Li XH, Xiao JH, Wang SP (2011) OsWRKY45 alleles play different roles in abscisic acid signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice. J Exp Bot 62:4863–4874

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tripathi P, Rabara RC, Langum TJ, Boken AK, Rushton DL, Boomsma DD, Rinerson CI, Rabara J, Reese RN, Chen X, Rohila JS, Rushton PJ (2012) The WRKY transcription factor family in Brachypodium distachyon. BMC Genom 13:270

    Article  CAS  Google Scholar 

  • Wang HH, Hao JJ, Chen XJ, Hao ZN, Wang X, Lou YG, Peng YL, Guo ZJ (2007) Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Mol Biol 65:799–815

    Article  CAS  PubMed  Google Scholar 

  • Wang C, Deng PY, Chen LL, Wang XT, Ma H, Hu W, Yao NC, Feng Y, Chai RH, Yang GX, He GY (2013) A wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco. PLoS One 8:e65120

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wen F, Zhu H, Li P, Jiang M, Mao WQ, Ong C, Chu ZQ (2014) Genome-wide evolutionary characterization and expression analyses of WRKY family genes in Brachypodium distachyon. DNA Res 21:327–339

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wu XL, Shiroto Y, Kishitani S, Ito Y, Toriyama K (2009) Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Rep 28(1):21–30

    Article  CAS  PubMed  Google Scholar 

  • Xiang JX, Ran J, Zou J, Zhou XY, Liu AL, Zhang XW, Peng Y, Tang N, Luo GY, Chen XB (2013) Heat shock factor OsHsfB2b negatively regulates drought and salt tolerance in rice. Plant Cell Rep 32:1795–1806

    Article  CAS  PubMed  Google Scholar 

  • Xie Z, Zhang ZL, Zou XL, Huang J, Ruas P, Thompson D, Shen QJ (2005) Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol 137:176–189

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xu XP, Chen CH, Fan BF, Chen ZX (2006) Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors. Plant Cell 18:1310–1326

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yang Q, Chen ZZ, Zhou XF, Yin HB, Li X, Xin XF, Hong XH, Zhu JK, Gong Z (2009) Overexpression of SOS (salt overly sensitive) genes increases salt tolerance in transgenic Arabidopsis. Mol Plant 2:22–31

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yang A, Dai XY, Zhang WH (2012) A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. J Exp Bot 63:2541–2556

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang CQ, Xu Y, Lu Y, Yu HX, Gu MH, Liu QQ (2011) The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice. Planta 234:541–554

    Article  CAS  PubMed  Google Scholar 

  • Zheng Z, Qamar SA, Chen Z, Mengiste T (2006) Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J 48:592–605

    Article  CAS  PubMed  Google Scholar 

  • Zhou QY, Tian AG, Zou HF, Xie ZM, Lei G, Huang J, Wang CM, Wang HW, Zhang JS, Chen SY (2008) Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnol J 6:486–503

    Article  CAS  PubMed  Google Scholar 

  • Zhu JK (2001) Cell signaling under salt, water and cold stresses. Curr Opin Plant Biol 4:401–406

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by International S & T Cooperation Key Projects of MoST (Grant no. 2009DFB30340), National Genetically Modified New Varieties of Major Projects of China (Grant no. 2014ZX08010004), Research Fund for the Doctoral Program of Higher Education of China (Grant no. 2012014211075) and Open Research Fund of State Key Laboratory of Hybrid Rice in Wuhan University (Grant no. KF201302). We thank Wuhan Botanic Garden of Chinese Academy of Sciences for supplying seeds of Brachypodium distachyon (Bd21).

Conflict of interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science & Technology, Wuhan, 430074, China

    Jiutong Sun, Wei Hu, Run Zhou, Lianzhe Wang, Xiatian Wang, Qiong Wang, Zhijuan Feng, Yaping Li, Ding Qiu, Guangyuan He & Guangxiao Yang

Authors
  1. Jiutong Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Run Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lianzhe Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiatian Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qiong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhijuan Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yaping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ding Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Guangyuan He
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Guangxiao Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Guangyuan He or Guangxiao Yang.

Additional information

Communicated by Leandro Peña.

J. Sun and W. Hu contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

299_2014_1684_MOESM1_ESM.tif

Alignment of BdWRKY36 protein sequence with the representative closely related proteins OsWRKY37 (DAA05102.1), OsWRKY66 (DAA05131.1), AtWRKY35 (NP_181029.1) and AtWR KY14 (NP_564359.1) using DNAMAN software The conserved WRKY domain with 60 amino acid residues, the putative nuclear localization signal with KRRK sequence and C-terminal region (from 200th aa to the end of C-terminus) of transcriptional activity were indicated by black lines. The cysteine and histidine residues of the putative zinc finger motif were marked by arrowheads (TIFF 1884 kb)

299_2014_1684_MOESM2_ESM.tif

The phylogenetic relationship between BdWRKY36 and other plant WRKY proteins. BdWRKY36 is shown in a frame. Numbers and letters on the right indicate different groups of WRKYs. Numbers above or below branches indicate bootstrap values (50 %) from 1,000 replicates. The protein sequences of the WRKYs used for construction of the tree are acquired from GenBank database The accession numbers are listed: OsWRKY37 (DAA05102.1), OsWRKY66 (DAA05131.1), OsWRKY43 (DAA05108.1), OsWRKY36 (DAA05101.1), OsWRKY45 (ABC02809.1), AtWRKY35 (NP_181029.1), AtWRKY14 (NP_564359.1), AtWRKY36 (NP_564976.1), AtWRKY72 (NP_197017.1), AtWRKY18 (NP_567882.1) AtWRKY40 (NP_178199.1), AtWRKY20 (NP_849450.1), AtWRKY34 (NP_194374.1), AtWRKY57 (NP_974112.1), AtWRKY28 (NP_193551.1), AtWRKY30 (NP_568439.1), AtWRKY55 (NP_001078033.1), AtWRKY17 (NP_565574.1), AtWRKY7 (NP_194155.1) (TIFF 251 kb)

299_2014_1684_MOESM3_ESM.tif

Time course RT-PCR analysis of expression profiles of BdWRKY36 under normal condition. Total RNA was isolated from leaves. BdUBC18 gene was used as an internal control. Two biological experiments were performed, which produced similar results (TIFF 426 kb)

Primers used for PCR analysis (DOCX 11 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, J., Hu, W., Zhou, R. et al. The Brachypodium distachyon BdWRKY36 gene confers tolerance to drought stress in transgenic tobacco plants. Plant Cell Rep 34, 23–35 (2015). https://doi.org/10.1007/s00299-014-1684-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-014-1684-6

Keywords

Search

Navigation