Abstract
Key message
The radish WRKY gene family was genome-widely identified and played critical roles in response to multiple abiotic stresses.
Abstract
The WRKY is among the largest transcription factors (TFs) associated with multiple biological activities for plant survival, including control response mechanisms against abiotic stresses such as heat, salinity, and heavy metals. Radish is an important root vegetable crop and therefore characterization and expression pattern investigation of WRKY transcription factors in radish is imperative. In the present study, 126 putative WRKY genes were retrieved from radish genome database. Protein sequence and annotation scrutiny confirmed that RsWRKY proteins possessed highly conserved domains and zinc finger motif. Based on phylogenetic analysis results, RsWRKYs candidate genes were divided into three groups (Group I, II and III) with the number 31, 74, and 20, respectively. Additionally, gene structure analysis revealed that intron–exon patterns of the WRKY genes are highly conserved in radish. Linkage map analysis indicated that RsWRKY genes were distributed with varying densities over nine linkage groups. Further, RT-qPCR analysis illustrated the significant variation of 36 RsWRKY genes under one or more abiotic stress treatments, implicating that they might be stress-responsive genes. In total, 126 WRKY TFs were identified from the R. sativus genome wherein, 35 of them showed abiotic stress-induced expression patterns. These results provide a genome-wide characterization of RsWRKY TFs and baseline for further functional dissection and molecular evolution investigation, specifically for improving abiotic stress resistances with an ultimate goal of increasing yield and quality of radish.
Similar content being viewed by others
Abbreviations
- aa:
-
Amino acids
- BLAST:
-
Basic local alignment search tool
- bp:
-
Base pair
- Cd:
-
Cadmium
- CDS:
-
Coding sequence
- GO:
-
Gene ontology
- HM:
-
Heavy metal
- LG:
-
Linkage group
- MW:
-
Molecular weight
- Pb:
-
Lead
- pI:
-
Isoelectric point
- RT-qPCR:
-
Reverse transcription-quantitative polymerase chain reaction
- TF:
-
Transcription factor
References
Bencke-Malato M, Cabreira C, Wiebke-Strohm B, Bücker-Neto L, Mancini E, Osorio MB, Homrich MS, Turchetto-Zolet AC, De Carvalho MC, Stolf R (2014) Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection. BMC Plant Biol 14:236
Cannon SB, Mitra A, Baumgarten A, Young ND, May G (2004) The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol 4:1–21
Chen H, Lai Z, Shi J, Xiao Y, Chen Z, Xu X (2010) Roles of Arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biol 10:281
Chen L, Song Y, Li S, Zhang L, Zou C, Yu D (2012) The role of WRKY transcription factors in plant abiotic stresses. Biochim Biophys Acta 1819:120–128
Chen L, Yang Y, Liu C, Zheng Y, Xu M, Wu N, Sheng J, Shen L (2015) Characterization of WRKY transcription factors in Solanum lycopersicum reveals collinearity and their expression patterns under cold treatment. Biochem Biophys Res Commun 464:962–968
Chu X, Wang C, Chen X, Lu W, Li H, Wang X, Hao L, Guo X (2016) Correction: the cotton WRKY gene GhWRKY41 positively regulates salt and drought stress tolerance in transgenic Nicotiana benthamiana. PLoS One 11:e0157026
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 Rep 68:81–92
Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676
Crooks GE, Hon G, Chandonia J-M, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190
Dai X, Wang Y, Zhang W-H (2015) OsWRKY74, a WRKY transcription factor, modulates tolerance to phosphate starvation in rice. J Exp Bot 67:947–960
Eulgem T, Rushton P, Robatzek S (2000) Somssich I (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206
Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, Lin J, Minguez P, Bork P, Von Mering C (2013) STRING v9. 1: protein–protein interaction networks, with increased coverage and integration. Nucleic Acids Res 41:D808–D815
Gautam S, Anjani K, Srivastava N (2016) In vitro evaluation of excess copper affecting seedlings and their biochemical characteristics in Carthamus tinctorius L. (variety PBNS-12). Physiol Mol Biol Plants 22:121–129
Goel R, Pandey A, Trivedi PK, Asif MH (2016) Genome-wide analysis of the Musa WRKY gene family: evolution and differential expression during development and stress. Front Plant Sci 7:209
Göhre V, Jones AM, Sklenář J, Robatzek S, Weber AP (2012) Molecular crosstalk between PAMP-triggered immunity and photosynthesis. Mol Plant Microbe Interact 25:1083–1092
Guo C, Guo R, Xu X, Gao M, Li X, Song J, Zheng Y, Wang X (2014) Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. J Exp Bot 65:1513–1528
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235
He H, Dong Q, Shao Y, Jiang H, Zhu S, Cheng B, Xiang Y (2012) Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa. Plant Cell Rep 31:1199–1217
He Y, Mao S, Gao Y, Zhu L, Wu D, Cui Y, Li J, Qian W (2016) Genome-wide identification and expression analysis of WRKY transcription factors under multiple stresses in Brassica napus. PLoS One 11:e0157558
Hsu F-C, Chou M-Y, Chou S-J, Li Y-R, Peng H-P, Shih M-C (2013) Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis. Plant Cell 25:2699–2713
Huh SU, Choi LM, Lee GJ, Kim YJ, Paek KH (2012) Capsicum annuum WRKY transcription factor d (CaWRKYd) regulates hypersensitive response and defense response upon Tobacco mosaic virus infection. Plant Sci 197:50–58
Jiang Y, Deyholos MK (2006) Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biol 6:1
Jiang Y, Duan Y, Yin J, Ye S, Zhu J, Zhang F, Lu W, Fan D, Luo K (2014) Genome-wide identification and characterization of the Populus WRKY transcription factor family and analysis of their expression in response to biotic and abiotic stresses. J Exp Bot 65(22):6629–6644
Joshi R, Wani SH, Singh B, Bohra A, Dar ZA, Lone AA, Pareek A, Singla-Pareek SL (2016) Transcription factors and plants response to drought stress: current understanding and future directions. Front Plant Sci 7:1029
Kitashiba H, Li F, Hirakawa H, Kawanabe T, Zou Z, Hasegawa Y, Tonosaki K, Shirasawa S, Fukushima A, Yokoi S (2014) Draft sequences of the radish (Raphanus sativus L.) genome. DNA Res 21:481–490
Kulhari A, Sheorayan A, Bajar S, Sarkar S, Chaudhury A, Kalia RK (2013) Investigation of heavy metals in frequently utilized medicinal plants collected from environmentally diverse locations of north western India. SpringerPlus 2:676
Lai Z, Li Y, Wang F, Cheng Y, Fan B, Yu J-Q, Chen Z (2011) Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense. Plant Cell 23:3824–3841
Letunic I, Copley RR, Pils B, Pinkert S, Schultz J, Bork P (2006) SMART 5: domains in the context of genomes and networks. Nucleic Acids Res 34:D257–D260
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
Li G, Meng X, Wang R, Mao G, Han L, Liu Y, Zhang S (2012) Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis. PLoS Genet 8:e1002767
Li H, Gao Y, Xu H, Dai Y, Deng D, Chen J (2013a) ZmWRKY33, a WRKY maize transcription factor conferring enhanced salt stress tolerances in Arabidopsis. J Plant Growth Regul 70:207–216
Li J, Besseau S, Törönen P, Sipari N, Kollist H, Holm L, Palva ET (2013b) Defense-related transcription factors WRKY70 and WRKY54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis. New Phytol 200:457–472
Ling J, Jiang W, Zhang Y, Yu H, Mao Z, Gu X, Huang S, Xie B (2011) Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genom 12:454
Liu RH, Meng J (2003) MapDraw: a microsoft excel macro for drawing genetic linkage maps based on given genetic linkage data. Yi Chuan 25:317–321
Liu S, Wang X, Wang H, Xin H, Yang X, Yan J, Li J, Tran L-SP, Shinozaki K, Yamaguchi-Shinozaki K (2013) Genome-wide analysis of ZmDREB genes and their association with natural variation in drought tolerance at seedling stage of Zea mays L. PLoS Genet 9:e1003790
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
Maeo K, Hayashi S, Kojima-Suzuki H, Morikami A, Nakamura K (2001) Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins. Biosci Biotechnol Biochem 65:2428–2436
Meng D, Li Y, Bai Y, Li M, Cheng L (2016) Genome-wide identification and characterization of WRKY transcriptional factor family in apple and analysis of their responses to waterlogging and drought stress. Plant Physiol Biochem 103:71–83
Mishra S, Triptahi V, Singh S, Phukan UJ, Gupta M, Shanker K, Shukla RK (2013) Wound induced tanscriptional regulation of benzylisoquinoline pathway and characterization of wound inducible PsWRKY transcription factor from Papaver somniferum. PLoS One 8:e52784
Mitsui Y, Shimomura M, Komatsu K, Namiki N, Shibata-Hatta M, Imai M, Katayose Y, Mukai Y, Kanamori H, Kurita K (2015) The radish genome and comprehensive gene expression profile of tuberous root formation and development. Sci Rep 5:10835
Mukhopadhyay M, Mondal TK (2015) Effect of zinc and boron on growth and water relations of Camellia sinensis. Natl Acad Sci Lett 38:283–286
Nie S, Xu L, Wang Y, Huang D, Muleke EM, Sun X, Wang R, Xie Y, Gong Y, Liu L (2015) Identification of bolting-related microRNAs and their targets reveals complex miRNA-mediated flowering-time regulatory networks in radish (Raphanus sativus L.). Sci Rep 5:14034
Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A (2009) The Sorghum bicolor genome and the diversification of grasses. Nature 457:551–556
Qiu Y, Yu D (2009) Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis. Environ Exp Bot 65:35–47
Rodriguez E, Santos C, Azevedo R, Moutinho-Pereira J, Correia C, Dias MC (2012) Chromium (VI) induces toxicity at different photosynthetic levels in pea. Plant Physiol Biochem 53:94–100
Ross C, Liu Y, Shen Q (2007) The WRKY gene family in rice (Oryza sativa). J Integr Plant Biol 49:827–842
Rushton P, Somssich I, Ringler P, Shen Q (2010) WRKY transcription factors. Trends Plant Sci 15:247–258
Saeed A, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J (2003) TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34:374–378
Scarpeci TE, Zanor MI, Mueller-Roeber B, Valle EM (2013) Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana. Plant Mol Biol 83:265–277
Schluttenhofer C, Yuan L (2015) Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol 167:295–306
Shaik R, Ramakrishna W (2013) Genes and co-expression modules common to drought and bacterial stress responses in Arabidopsis and rice. PLoS One 8:e77261
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504
Shen D, Sun H, Huang M, Zheng Y, Qiu Y, Li X, Fei ZJ (2013) Comprehensive analysis of expressed sequence tags from cultivated and wild radish (Raphanus spp.). BMC Genom 14:721
Song X, Li Y, Hou X (2013) Genome-wide analysis of the AP2/ERF transcription factor superfamily in Chinese cabbage (Brassica rapa ssp. pekinensis). BMC Genom 14:1
Sun X, Xu L, Wang Y, Yu R, Zhu X, Luo X, Gong Y, Wang R, Limera C, Zhang K, Liu L (2015a) Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (Raphanus sativus L.). BMC Genom 16:197
Sun Y, Qiu Y, Zhang X, Chen X, Shen D, Wang H, Li X (2015b) Genome-wide identification of microRNAs associated with taproot development in radish (Raphanus sativus L.). Gene 569:118–126
Sun X, Xu L, Wang Y, Luo X, Zhu X, Kinuthia KB, Nie S, Feng H, Li C, Liu L (2016) Transcriptome-based gene expression profiling identifies differentially expressed genes critical for salt stress response in radish (Raphanus sativus L.). Plant Cell Rep 35:329–346
Tang J, Wang F, Wang Z, Huang Z, Xiong A, Hou X (2013) Characterization and co-expression analysis of WRKY orthologs involved in responses to multiple abiotic stresses in Pak-choi (Brassica campestris ssp. chinensis). BMC Plant Biol 13:1
Teige M, Scheikl E, Eulgem T, Dóczi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15:141–152
Vernay P, Gauthier-Moussard C, Hitmi A (2007) Interaction of bioaccumulation of heavy metal chromium with water relation, mineral nutrition and photosynthesis in developed leaves of Lolium perenne L. Chemosphere 68:1563–1575
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
Wang Y, Xu L, Chen Y, Shen H, Gong Y, Limera C, Liu L (2013) Transcriptome profiling of radish (Raphanus sativus L.) root and identification of genes involved in response to Lead (Pb) stress with next generation sequencing. PLoS One 8:e66539
Wang L, Zhu W, Fang L, Sun X, Su L, Liang Z, Wang N, Londo JP, Li S, Xin H (2014) Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera. BMC Plant Biol 14:1
Wang M, Vannozzi A, Wang G, Zhong Y, Corso M, Cavallini E, Cheng Z-MM (2015a) A comprehensive survey of the grapevine VQ gene family and its transcriptional correlation with WRKY proteins. Front Plant Sci 6:417
Wang R, Xu L, Zhu X, Zhai L, Wang Y, Yu R, Gong Y, Limera C, Liu L (2015b) Transcriptome-wide characterization of novel and heat-stress-responsive microRNAs in radish (Raphanus sativus L.) using next-generation sequencing. Plant Mol Biol Rep 33:867–880
Wang Z, Tang J, Hu R, Wu P, Hou XL, Song XM, Xiong AS (2015c) Genome-wide analysis of the R2R3-MYB transcription factor genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveals their stress and hormone responsive patterns. BMC Genom 16:17
Wei W, Zhang Y, Han L, Guan Z, Chai T (2008) A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco. Plant Cell Rep 27:795–803
Wei KF, Chen J, Chen YF, Wu LJ, Xie DX (2012) Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize. DNA Res 19:153–164
Wu Z-J, Li X-H, Liu Z-W, Li H, Wang Y-X, Zhuang J (2015) Transcriptome-based discovery of AP2/ERF transcription factors related to temperature stress in tea plant (Camellia sinensis). Funct Integr Genom 15:741–752
Xie Y, Ye S, Wang Y, Xu L, Zhu X, Yang J, Feng H, Yu R, Karanja B, Gong Y (2015) Transcriptome-based gene profiling provides novel insights into the characteristics of radish root response to Cr stress with next-generation sequencing. Front Plant Sci 6:202
Xu L, Wang Y, Xu Y, Wang L, Zhai L, Zhu X, Gong Y, Ye S, Liu L (2013a) Identification and characterization of novel and conserved microRNAs in radish (Raphanus sativus L.) using high-throughput sequencing. Plant Sci 201–202:108–114
Xu L, Wang Y, Zhai LL, Xu Y, Wang LJ, Zhu XW, Gong YQ, Yu RG, Limera C, Liu LW (2013b) Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots. J Exp Bot 64:4271–4287
Yang G, Wang C, Wang Y, Guo Y, Zhao Y, Yang C, Gao C (2016) Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of cadmium stress. Sci Rep 6:18752
Yu R, Wang Y, Xu L, Zhu X, Zhang W, Wang R, Gong Y, Limera C, Liu L (2015) Transcriptome profiling of root microRNAs reveals novel insights into taproot thickening in radish (Raphanus sativus L.). BMC Plant Biol 15:30
Zentgraf U, Laun T, Miao Y (2010) The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana. Eur J Cell Biol 89:133–137
Zhai L, Xu L, Wang Y, Zhu X, Feng H, Li C, Luo X, Everlyne MM, Liu L (2016) Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.). Sci Rep 6:21652
Zhang H, Jin J, Tang L, Zhao Y, Gu X, Gao G, Luo J (2011) PlantTFDB 2.0: update and improvement of the comprehensive plant transcription factor database. Nucleic Acids Res 39:D1114–D1117
Zhang W, Xie Y, Xu L, Wang Y, Zhu X, Wang R, Zhang Y, Muleke E, Liu L (2016) Identification of microRNAs and their target genes explores miRNA-mediated regulatory network of cytoplasmic male sterility occurrence during anther development in radish (Raphanus sativus L.). Front. Plant Sci 7:1054
Zhao H, Wu L, Chai T, Zhang Y, Tan J, Ma S (2012) The effects of copper, manganese and zinc on plant growth and elemental accumulation in the manganese-hyperaccumulator Phytolacca americana. J Plant Physiol 169:1243–1252
Zhao H, Wang S, Chen S, Jiang J, Liu G (2015) Phylogenetic and stress-responsive expression analysis of 20 WRKY genes in Populus simonii × Populus nigra. Gene 565:130–139
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
Acknowledgements
The current work was partly funded by Grants from the Natural Science Foundation of China (31372064, 31501759, 31601766), National Key Technology Research and Development Program of China (2016YFD0100204-25), Key Technology R&D Program of Jiangsu Province (BE2016379), and Jiangsu Agricultural Science and Technology Innovation Fund (CX(16)1012).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Qiaochun Wang.
Electronic supplementary material
Below is the link to the electronic supplementary material.
299_2017_2190_MOESM1_ESM.tif
Fig. S1 Putative isoelectric points and molecular weights of WRKY proteins Group I, II and III in R. sativus (TIFF 248 kb)
299_2017_2190_MOESM2_ESM.tif
Fig. S2 The schematic diagram of the logo diagrams of ten motifs analyzed in all the 126 WRKY protein sequence in radish (TIFF 497 kb)
299_2017_2190_MOESM3_ESM.tif
Fig. S3 Gene ontology (GO) analysis and distribution of 126 RsWRKY genes into biological process, cellular component, and molecular functions (TIFF 410 kb)
299_2017_2190_MOESM4_ESM.tif
Fig. S4 Heat map showing WRKY genes expression pattern in radish expressed at various developmental stages in leaves, root, root tips, and cambium of radish. The heat map was created using Multi-Experiment Viewer (MeV 4.8) program (TIFF 2064 kb)
299_2017_2190_MOESM5_ESM.tif
Fig. S5 Heat map illustrating WRKY gene transcripts pattern from our radish lab data at different growth stages and biotic stresses. The heat map was created using Multi-Experiment Viewer (MeV 4.8) program (TIFF 1381 kb)
Rights and permissions
About this article
Cite this article
Karanja, B.K., Fan, L., Xu, L. et al. Genome-wide characterization of the WRKY gene family in radish (Raphanus sativus L.) reveals its critical functions under different abiotic stresses. Plant Cell Rep 36, 1757–1773 (2017). https://doi.org/10.1007/s00299-017-2190-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-017-2190-4