Skip to main content
Log in

Functional analysis of a cold-responsive rice WRKY gene, OsWRKY71

  • Original Article
  • Published:
Plant Biotechnology Reports Aims and scope Submit manuscript

Abstract

Publicly available microarray data and RNA gel blot analysis identified a rice WRKY transcription factor, OsWRKY71, that is highly upregulated in response to cold stress. Experiments with OsWRKY71 promoter:GFP transgenic rice confirmed its cold-inducible expression. Transient expression of OsWRKY71-GFP in maize mesophyll protoplasts indicated that it is localized predominantly in the nucleus and to a lesser extent in the cytosol. Transcriptional activation assays revealed that OsWRKY71 suppresses luciferase reporter activity in maize protoplasts, suggesting that it functions as a transcriptional repressor in rice. To characterize the function of OsWRKY71 in rice, we generated transgenic rice plants carrying CaMV35S promoter:OsWRKY71. Upon cold (4 °C) treatment, two selected OsWRKY71 transgenic lines, OX12 and OX21, recovered much better with respect to survival rate, photosynthetic ability, fresh weight, and dry weight than the control lines. RT-PCR analysis of known cold-responsive genes found that expression of OsTGFR and WSI76 was increased in OsWRKY71 transgenic lines in response to cold stress. Our results suggest that OsWRKY71 has a positive function in cold tolerance by regulating downstream target genes.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Amir Hossain MA, Cho JI, Han M, Ahn CH, Jeon JS, An G, Park PB (2010) The ABRE-binding bZIP transcription factor OsABF2 is a positive regulator of abiotic stress and ABA signaling in rice. J Plant Physiol 167:1512–1520

    Article  CAS  Google Scholar 

  • Andaya VC, Mackill DJ (2003) Mapping of QTLs associated with cold tolerance during the vegetative stage in rice. J Exp Bot 54:2579–2585

    Article  PubMed  CAS  Google Scholar 

  • Badawi M, Danyluk J, Boucho B, Houde M, Sarhan F (2007) The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs. Mol Genet Genom 277:533–554

    Article  CAS  Google Scholar 

  • Baruah A, Ishigo-Oka N, Adachi M, Oguma Y, Tokizono Y, Onishi K, Sano Y (2009) Cold tolerance at the early growth stage in wild and cultivated rice. Euphytica 165:459–470

    Article  Google Scholar 

  • Birkenbihl RP, Diezel C, Somssich IE (2012) Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection. Plant Physiol 159:266–285

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Cai M, Qiu D, Yuan T, Ding X, Li H, Duan L, Xu C, Li X, Wang S (2008) Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance. Plant Cell Environ 31:86–96

    Article  PubMed  CAS  Google Scholar 

  • Casaretto J, Ho TH (2003) The transcription factors HvABI5 and HvVP1 are required for the abscisic acid induction of gene expression in barley aleurone cells. Plant Cell 15:271–284

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Chen JQ, Meng XP, Zhang Y, Xia M, Wang XP (2008) Over-expression of OsDREB genes lead to enhanced drought tolerance in rice. Biotechnol Lett 30:2191–2198

    Article  PubMed  CAS  Google Scholar 

  • Chen L, Song Y, Li S, Zhang L, Zou C, Yu D (2012) The role of WRKY transcription factors in plant abiotic stresses. BBA-Gene Regul 1819:120–128

    CAS  Google Scholar 

  • Cheng C, Yun KY, Ressom HW, Mohanty B, Bajic VB, Jia Y, Yun SJ, de los Reyes BG (2007) An early response regulatory cluster induced by low temperature and hydrogen peroxide in seedlings of chilling-tolerant japonica rice. BMC Genom 8:175

    Article  CAS  Google Scholar 

  • Cho JI, Ryoo N, Ko S, Lee SK, Lee J, Jung KH, Lee YH, Bhoo SH, Winderickx J, An G et al (2006) Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.). Planta 224:598–611

    Article  PubMed  CAS  Google Scholar 

  • Cho JI, Ryoo N, Eom JS, Lee DW, Kim HB, Jeong SW, Lee YH, Kwon YK, Cho MH, Bhoo SH et al (2009) Role of the rice hexokinases OsHXK5 and OsHXK6 as glucose sensors. Plant Physiol 149:745–759

    Article  PubMed Central  PubMed  Google Scholar 

  • Chujo T, Kato T, Yamada K, Takai R, Akimoto-Tomiyama C, Minami E, Nagamura Y, Shibuya N, Yasuda M, Nakashita H et al (2008) Characterization of an elicitor-induced rice WRKY gene, OsWRKY71. Biosci Biotech Biochem 72:240–245

    Article  CAS  Google Scholar 

  • Chujo T, Miyamoto K, Shimogawa T, Shimizu T, Otake Y, Yokotani N, Nishizawa Y, Shibuya N, Nojiri H, Yamane H et al (2013) OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus. Plant Mol Biol 82:23–37

    Article  PubMed  CAS  Google Scholar 

  • Ciolkowski I, Wanke D, Birkenbihl R, Somssich I (2008) Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY domain function. Plant Mol Biol 68:81–92

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Claes B, Dekeyser R, Villarroel R, Van den Bulcke M, Bauw G, Van Montagu M, Caplan A (1990) Characterization of a rice gene showing organ-specific expression in response to salt stress and drought. Plant Cell 2:19–27

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Dang FF, Wang YN, Yu L, Eulgem T, Lai Y, Liu ZQ, Wang X, Qiu AL, Zhang TX, Lin J et al (2012) CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant Cell Environ 36:757–774

    Article  PubMed  CAS  Google Scholar 

  • de Los Reyes BG, Yun SJ, Herath V, Xu F, Park MR, Lee JI, Kim KY (2013) Phenotypic, physiological, and molecular evaluation of rice chilling stress response at the vegetative stage. Methods Mol Biol 956:227–241

    Article  PubMed  CAS  Google Scholar 

  • Delteil A, Blein M, Faivre-Rampant O, Guellim A, Estevan J, Hirsch J, Bevitori R, Michel C, Morel JB (2012) Building a mutant resource for the study of disease resistance in rice reveals the pivotal role of several genes involved in defence. Mol Plant Pathol 13:72–82

    Article  PubMed  CAS  Google Scholar 

  • Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Fowler SG, 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 14:1675–1690

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Fowler SG, Cook D, Thomashow MF (2005) Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol 137:961–968

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Fujita Y, Fujita M, Satoh R, Maruyama K, Parvez MM, Seki M, Hiratsu K, Ohme-Takagi M, Shinozaki K, Yamaguchi-Shinozaki K (2005) AREB1 is a transcription activator of novel ABRE-dependent ABA-signaling that enhances drought stress tolerance in Arabidopsis. Plant Cell 17:3470–3488

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92

    Article  CAS  Google Scholar 

  • Han M, Ryu HS, Kim CY, Park DS, Ahn YK, Jeon JS (2013) OsWRKY30 is a transcription activator that enhances rice resistance to the Xanthomonas oryzae pathovar oryzae. J Plant Biol 56:258–265

    Article  CAS  Google Scholar 

  • Hu H, You J, Fang Y, Zhu X, Qi Z, Xiong L (2008) Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant Mol Biol 67:169–181

    Article  PubMed  CAS  Google Scholar 

  • Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153

    Article  PubMed  CAS  Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651

    Article  PubMed  CAS  Google Scholar 

  • Jeon JS, Lee S, Jung KH, Jun SH, Jeong DH, Lee J, Kim C, Jang S, Yang K, Nam J et al (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J 22:561–570

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Jiang CJ, Shimono M, Sugano S, Kojima M, Yazawa K, Yoshida R, Inoue H, Hayashi N, Sakakibara H, Takatsuji H (2010) Abscisic acid interacts antagonistically with salicylic acid signaling pathway in rice–Magnaporthe grisea interaction. Mol Plant Microbe Interact 23:791–798

    Article  PubMed  CAS  Google Scholar 

  • Karimi M, Inze D, Depicker A (2002) Gateway vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

  • Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basic. Ann Rev Plant Physiol Plant Mol Biol 42:313–349

    Article  CAS  Google Scholar 

  • Lee SC, Huh KW, An K, An G, Kim SR (2004) Ectopic expression of a cold-inducible transcription factor, CBF1/DREB1b, in transgenic rice (Oryza sativa L.). Mol Cells 18:107–114

    PubMed  CAS  Google Scholar 

  • Li S, Fu Q, Chen L, Huang W, Yu D (2011) Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta 233:1237–1252

    Article  PubMed  CAS  Google Scholar 

  • Liu XQ, Bai XQ, Wang XJ, Chu CC (2007) OsWRKY71, a rice transcription factor, is involved in rice defense response. J Plant Physiol 164:969–979

    Article  PubMed  CAS  Google Scholar 

  • Ma Q, Dai X, Xu Y, Guo J, Liu Y, Chen N, Xiao J, Zhang D, Xu Z, Zhang X et al (2009) Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes. Plant Physiol 150:244–256

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Maruyama K, Sakuma Y, Kasuga M, Ito Y, Seki M, Goda H, Shimada Y, Yoshida S, Shinozaki K, Yamaguchi-Shinozaki K (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J 38:982–993

    Article  PubMed  CAS  Google Scholar 

  • Matsushita A, Inoue H, Goto S, Nakayama A, Sugano S, Hayashi N, Takatsuji H (2012) The nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program. Plant J 73:302–313

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Mittal D, Madhyastha DA, Grover A (2012) Genome-wide transcriptional profiles during temperature and oxidative stress reveal coordinated expression patterns and overlapping regulons in rice. PLoS One 7:e40899

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51:617–630

    Article  PubMed  CAS  Google Scholar 

  • Ohta M, Ohme-Takagi M, Shinshi H (2000) Three ethylene-responsive transcription factors in tobacco with distinct transactivation functions. Plant J 22:29–38

    Article  PubMed  CAS  Google Scholar 

  • Park SH, Yi N, Kim YS, Jeong MH, Bang SW, Choi YD, Kim JK (2010) Analysis of five novel putative constitutive gene promoters in transgenic rice plants. J Exp Bot 61:2459–2467

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Peng Y, Bartley LE, Chen X, Dardick C, Chern M, Ruan R, Canlas PE, Ronald PC (2008) OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice. Mol Plant 1:446–458

    Article  PubMed  CAS  Google Scholar 

  • Peng Y, Bartley LE, Canlas PE, Ronald PC (2010) OsWRKY IIa transcription factors modulate rice innate immunity. Rice 3:36–42

    Article  PubMed Central  PubMed  Google Scholar 

  • Peng X, Zhang L, Zhang L, Liu Z, Cheng L, Yang Y, Shen S, Chen S (2013) The transcriptional factor LcDREB2 cooperates with LcSAMDC2 to contribute to salt tolerance in Leymus chinensis. Plant Cell Tiss Org Cult 113:245–256

    Article  CAS  Google Scholar 

  • Pramanik MH, Imai R (2005) Functional identification of a trehalose 6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice. Plant Mol Biol 58:751–762

    Article  PubMed  CAS  Google Scholar 

  • Rice WRKY Working Group (2012) Nomenclature report on rice WRKY’s—conflict regarding gene names and its solution. Rice 5:1–3

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Saito M, Yoshida M (2011) Expression analysis of the gene family associated with raffinose accumulation in rice seedlings under cold stress. J Plant Physiol 168:2268–2271

    Article  PubMed  CAS  Google Scholar 

  • Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31:279–292

    Article  PubMed  CAS  Google Scholar 

  • Seo YS, Chern M, Bartley LE, Han M, Jung KH, Le I, Walia H, Richter T, Xu X, Cao P et al (2011) Towards establishment of a rice stress response interactome. PLoS Genet 7:e1002020

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Shen QH, Saijo Y, Mauch S, Biskup C, Bieri S, Keller B, Seki H, Ulker B, Somssich IE, Schulze-Lefert P (2007) Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 315:1098–1103

    Article  PubMed  CAS  Google Scholar 

  • Shimono M, Sugano S, Nakayama A, Jiang CJ, Ono K, Toki S, Takatsuji H (2007) Rice WRKY45 plays a crucial role in benzothiazole-inducible blast resistance. Plant Cell 19:2064–2076

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Shimono H, Ishii A, Kanda E, Suto M, Nagano K (2011a) Genotypic variation in rice cold tolerance responses during reproductive growth as a function of water temperature during vegetative growth. Crop Sci 51:290–297

    Article  Google Scholar 

  • Shimono M, Koga H, Akagi A, Hayashi N, Goto S, Sawada M, Kurihara T, Matsushita A, Sugano S, Jiang CJ et al (2011b) Rice WRKY45 plays important roles in fungal and bacterial disease resistance. Mol Plant Pathol 13:83–94

    Article  PubMed  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (1996) Molecular responses to drought and cold stress. Curr Opin Biotech 7:161–167

    Article  PubMed  CAS  Google Scholar 

  • Singh KB, Foley RC, Oñate-Sánchez L (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5:430–436

    Article  PubMed  CAS  Google Scholar 

  • Skinner JS, von Zitzewitz J, Szucs P, Marquez-Cedillo L, Filichkin T, Amundsen K, Stockinger EJ, Thomashow MF, Chen TH, Hayes PM (2005) Structural, functional, and phylogenetic characterization of a large CBF gene family in barley. Plant Mol Biol 59:533–551

    Article  PubMed  CAS  Google Scholar 

  • Su CF, Wang YC, Hsieh TH, Lu CA, Tseng TH, Yu SM (2010) A novel MYBS3-dependent pathway confers cold tolerance in rice. Plant Physiol 153:145–158

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29:417–426

    Article  PubMed  CAS  Google Scholar 

  • Ulker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7:491–498

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Liu S, Liu X, Chen Z, Liu X, Pang Y, Sun X, Tang K (2004) Molecular cloning and characterization of a CBF gene from Capsella bursapastoris. DNA Seq 15:180–187

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Hao J, Chen X, Hao Z, Wang X, Lou Y, Peng Y, Guo Z (2007) Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Mol Biol 65:799–815

    Article  PubMed  CAS  Google Scholar 

  • Wang Z, Zhu Y, Wang L, Liu X, Liu Y, Phillips J, Deng X (2009) A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter. Planta 230:1155–1166

    Article  PubMed  CAS  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  PubMed Central  PubMed  CAS  Google Scholar 

  • Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57:781–803

    Article  PubMed  CAS  Google Scholar 

  • Yang A, Dai X, 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  PubMed Central  PubMed  CAS  Google Scholar 

  • Yokotani N, Sato Y, Tanabe S, Chujo T, Shimizu T, Okada K, Yamane H, Shimono M, Sugano S, Takatsuji H et al (2013) WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance. J Exp Bot 64:5085–5097

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Zhang ZL, Xie Z, Zou X, Casaretto J, Ho TH, Shen QJ (2004) A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol 134:1500–1513

    Article  PubMed Central  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Zhang T, Zhao X, Wang W, Pan Y, Huang L, Liu X, Zong Y, Zhu L, Yang D, Fu B (2012) Comparative transcriptome profiling of chilling stress responsiveness in two contrasting rice genotypes. PLoS One 7:e43274

    Article  PubMed Central  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Zou CS, Jiang WB, Yu DQ (2010) Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis. J Exp Bot 61:3901–3914

    Article  PubMed Central  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Mid-Career Researcher Program (NRF-2013R1A2A2A01068887 to J.-S. Jeon) and Basic Science Research Program (NRF-2014R1A1A1003903 to S.-K. Lee) of the National Research Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jong-Seong Jeon.

Additional information

C.-Y. Kim, K. T. X. Vo, and C. D. Nguyen have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 121 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, CY., Vo, K.T.X., Nguyen, C.D. et al. Functional analysis of a cold-responsive rice WRKY gene, OsWRKY71 . Plant Biotechnol Rep 10, 13–23 (2016). https://doi.org/10.1007/s11816-015-0383-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11816-015-0383-2

Keywords

Navigation