Alfalfa (Medicago sativa L.) is the most widely cultivated leguminous herb in the world. Its agricultural development has been restricted by various adverse environmental conditions, including water deficiency, high salinity, and low temperature. WRKY transcription factors (TFs) serve important roles in the regulation of plant development and stress responses. Research on the WRKY gene family has been reported for several species, but minimal information is available for alfalfa. In the present study, a total of 107 WRKY genes were identified in alfalfa and divided into 3 main groups. The classification, evolution, conserved motifs, and tissue expression were comprehensively analyzed. Meanwhile, 27 MsWRKY candidate genes that may be involved in abiotic stress were isolated through an analysis of gene expression profiles under different stresses, including cold, abscisic acid, drought, and salt treatments. Additionally, investigation of the cis-elements and potential biological functions of these genes further revealed that MsWRKY TFs may serve important roles in multiple stress resistance in alfalfa. This study provides an important foundation for future cloning and functional studies of WRKY genes in alfalfa.
Get full access to this article
View all access options for this article.
References
1.
AliM.A., AzeemF., NawazM.A., AcetT., AbbasA., ImranQ.M., et al. (2018). Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis. J Plant Physiol, 226, 12–21.
2.
AltschulS.F., GishW., MillerW., MyersE.W., and LipmanD.J. (1990). Basic local alignment search tool. J Mol Biol, 215, 403–410.
3.
BaileyT.L., BodenM., BuskeF.A., FrithM., GrantC.E., ClementiL., et al. (2009). MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res, 37, W202–W208.
4.
CamachoC., CoulourisG., AvagyanV., MaN., PapadopoulosJ., BealerK., et al. (2009). BLAST+: architecture and applications. BMC Bioinformatics 10,421.
5.
ChanwalaJ., SatpatiS., DixitA., ParidaA., GiriM.K., and DeyN. (2020). Genome-wide identification and expression analysis of WRKY transcription factors in pearl millet (Pennisetum glaucum) under dehydration and salinity stress. BMC Genomics, 21, 1–16.
6.
ChenC., ChenH., ZhangY., ThomasH.R., FrankM.H., HeY., et al. (2020). TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant, 13, 1194–1202.
7.
ChenL., XiangS., ChenY., LiD., and YuD. (2017). Arabidopsis WRKY45 interacts with the DELLA protein RGL1 to positively regulate age-triggered leaf senescence. Mol Plant, 10, 1174–1189.
8.
DollJ., MuthM., RiesterL., NebelS., BressonJ., LeeH.C., et al. (2019). Arabidopsis thaliana WRKY25 transcription factor mediates oxidative stress tolerance and regulates senescence in a redox-dependent manner. Front Plant Sci 10,1734.
9.
DongJ.X., ChenC.H., and ChenZ.X. (2003). Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol, 51, 21–37.
10.
El-GebaliS., MistryJ., BatemanA., EddyS.R., LucianiA., PotterS.C., et al. (2018). The Pfam protein families database in 2019. Nucleic Acids Res, 47, D427–D432.
11.
EmanuelssonO., NielsenH., BrunakS., and Von HeijneG. (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol, 300, 1005–1016.
12.
EulgemT., RushtonP.J., RobatzekS., and SomssichI.E. (2000). The WRKY superfamily of plant transcription factors. Trends Plant Sci, 5, 199–206.
13.
FranceschiniA., SzklarczykD., FrankildS., KuhnM., SimonovicM., RothA., et al. (2012). STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res, 41, D808–D815.
14.
GasteigerE., GattikerA., HooglandC., IvanyiI., AppelR.D., and BairochA. (2003). ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res, 31, 3784–3788.
15.
GilbertM.E., and MedinaV. (2016). Drought adaptation mechanisms should guide experimental design. Trends Plant Sci, 21, 639–647.
16.
HeG.H., XuJ.Y., WangY.X., LiuJ.M., LiP.S., ChenM., et al. (2016). Drought-responsive WRKY transcription factor genes TaWRKY1 and TaWRKY33 from wheat confer drought and/or heat resistance in Arabidopsis. BMC Plant Biol 16,116.
17.
HortonP., ParkK.J., ObayashiT., FujitaN., HaradaH., Adams-CollierC.J., et al. (2007). WoLF PSORT: protein localization predictor. Nucleic Acids Res, 35, W585–W587.
18.
HuZ., WangR., ZhengM., LiuX., MengF., WuH., et al. (2018). TaWRKY51 promotes lateral root formation through negative regulation of ethylene biosynthesis in wheat (Triticum aestivum L.). Plant J, 96, 372–388.
19.
HuangS.X., GaoY.F., LiuJ.K., PengX.L., NiuX.L., FeiZ.J., et al. (2012). Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum. Mol Genet Genomic, 287, 495–513.
20.
IshiguroS., and NakamuraK. (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.
21.
JiangJ., MaS., YeN., JiangM., CaoJ., and ZhangJ. (2017). WRKY transcription factors in plant responses to stresses. J Integr Plant Biol, 59, 86–101.
22.
JiangY., and DeyholosM.K. (2009). Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol, 69, 91–105.
23.
JiaoZ., SunJ., WangC., DongY., XiaoS., GaoX., et al. (2018). Genome-wide characterization, evolutionary analysis of WRKY genes in Cucurbitaceae species and assessment of its roles in resisting to powdery mildew disease. PLoS One 13,e0199851.
24.
JinJ., TianF., YangD.C., MengY.Q., KongL., LuoJ., et al. (2017). PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res, 45,(D1),D1040–D1045.
25.
JinX., YinX., NdayambazaB., ZhangZ., MinX., LinX., et al. (2019). Genome-wide identification and expression profiling of the ERF gene family in Medicago sativa L. under various abiotic stresses. DNA Cell Biol, 38, 1056–1068.
26.
KelleyL.A., MezulisS., YatesC.M., WassM.N., and SternbergM.J. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc, 10, 845–858.
27.
KumarS., StecherG., and TamuraK. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 33, 1870–1874.
28.
LescotM., DéhaisP., ThijsG., MarchalK., MoreauY., Van de PeerY., et al. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 30, 325–327.
29.
LiH., JiangF., WuP., WangK., and CaoY. (2020a). A high-quality genome sequence of model legume lotus japonicus (MG-20) provides insights into the evolution of root nodule symbiosis. Genes 11,483.
30.
LiS., FuQ., ChenL., HuangW., and YuD. (2011). Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta, 233, 1237–1252.
31.
LiW., and GodzikA. (2006). Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics, 22, 1658–1659.
32.
LiW., WangH., and YuD. (2016). Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions. Mol Plant, 9, 1492–1503.
33.
LiX., GuoW., LiJ., YueP., BuH., JiangJ., et al. (2020b). Histone acetylation at the promoter for the transcription factor PuWRKY31 affects sucrose accumulation in pear fruit. Plant Physiol, 182, 2035–2046.
34.
LiuL., ZhangZ., DongJ., and WangT. (2016a). Overexpression of MtWRKY76 increases both salt and drought tolerance in Medicago truncatula. Environ Exp Bot, 123, 50–58.
35.
LiuW., XiongC., YanL., ZhangZ., MaL., WangY., et al. (2017). Transcriptome analyses reveal candidate genes potentially involved in Al stress response in alfalfa. Front Plant Sci 8,26.
36.
LiuW., ZhangZ., ChenS., MaL., WangH., DongR., et al. (2016b). Global transcriptome profiling analysis reveals insight into saliva-responsive genes in alfalfa. Plant Cell Rep, 35, 561–571.
37.
LiuZ., ChenT., MaL., ZhaoZ., ZhaoP.X., NanZ., et al. (2013). Global transcriptome sequencing using the Illumina platform and the development of EST-SSR markers in autotetraploid alfalfa. PLoS One 8,e83549.
38.
LivakK.J., and SchmittgenT.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−△△Ct method. Methods, 25, 402–408.
39.
LlorcaC.M., PotschinM., and ZentgrafU. (2014). bZIPs and WRKYs: two large transcription factor families executing two different functional strategies. Front Plant Sci 5,169.
40.
LuoD., WuY., LiuJ., ZhouQ., LiuW., WangY., et al. (2019a). Comparative transcriptomic and physiological analyses of Medicago sativa L. indicates that multiple regulatory networks are activated during continuous ABA treatment. Int J Mol Sci 20,47.
41.
LuoD., ZhouQ., WuY., ChaiX., LiuW., WangY., et al. (2019b). Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.). BMC Plant Biol 19,32.
42.
MaeoK., HayashiS., Kojima-SuzukiH., MorikamiA., and NakamuraK. (2001). Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins. Biosci Biotech Bioch, 65, 2428–2436.
43.
NakashimaK., ItoY., and Yamaguchi-ShinozakiK. (2009). Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol, 149, 88–95.
44.
NakashimaK., Yamaguchi-ShinozakiK., and ShinozakiK. (2014). The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat. Front Plant Sci 5,170.
45.
NanH., and GaoL.Z. (2019). Genome-wide analysis of WRKY genes and their response to hormone and mechanic stresses in carrot. Front Genet 10,363.
46.
O'RourkeJ.A., FuF., BucciarelliB., YangS.S., SamacD.A., LambJ.F., et al. (2015). The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies. BMC Genomics 16,502.
47.
PuranikS., SahuP.P., MandalS.N., ParidaS.K., and PrasadM. (2013). Comprehensive genome-wide survey, genomic constitution and expression profiling of the NAC transcription factor family in foxtail millet (Setaria italica L.). PLoS One 8,e64594.
48.
RushtonP.J., MacdonaldH., HuttlyA.K., LazarusC.M., and HooleyR. (1995). Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of α-Amy2 genes. Plant Mol Biol, 29, 691–702.
RushtonP.J., TorresJ.T., ParniskeM., WernertP., HahlbrockK., and SomssichI.E. (1996). Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J, 15, 5690–5700.
51.
SantnerA., and EstelleM. (2009). Recent advances and emerging trends in plant hormone signaling. Nature, 459, 1071–1078.
52.
SmýkalP., von WettbergE.J.B, and McPheeK. (2020). Legume genetics and biology: from Mendel's pea to legume genomics. Int J Mol Sci 21,3336.
53.
SongG., LiX., MunirR., KhanA.R., AzharW., YasinM.U., et al. (2020). The WRKY6 transcription factor affects seed oil accumulation and alters fatty acid compositions in Arabidopsis thaliana. Physiol Plantarum, 169, 612–624.
54.
SongH., and NanZ. (2014). Genome-wide identification and analysis of WRKY transcription factors in Medicago truncatula. Hereditas, 36, 152–168.
55.
SongH., WangP., NanZ., and WangX. (2014). The WRKY transcription factor genes in Lotus japonicus. Int J Genomics 2014,420128.
56.
WangC.T., RuJ.N., LiuY.W., YangJ.F., LiM., XuZ.S., et al. (2018a). The maize WRKY transcription factor ZmWRKY40 confers drought resistance in transgenic Arabidopsis. Int J Mol Sci 19,2580.
57.
WangY., JiangL., ChenJ., TaoL., AnY., CaiH., et al. (2018b) Overexpression of the alfalfa WRKY11 gene enhances salt tolerance in soybean. PLoS One 13, e0192382.
58.
WaqasM., AzharM.T., RanaI.A., AzeemF., AliM.A., NawazM.A., et al. (2019). Genome-wide identification and expression analyses of WRKY transcription factor family members from chickpea (Cicer arietinum L.) reveal their role in abiotic stress-responses. Genes Genom, 41, 467–481.
59.
WeiW., LiangD.W., BianX.H., ShenM., XiaoJ.H., ZhangW.K., et al. (2019). GmWRKY54 improves drought tolerance through activating genes in abscisic acid and Ca2+ signaling pathways in transgenic soybean. Plant J, 100, 384–398.
60.
WuG.Q., LiZ.Q., CaoH., and WangJ.L. (2019). Genome-wide identification and expression analysis of the WRKY genes in sugar beet (Beta vulgaris L.) under alkaline stress. PeerJ 7,e7817.
61.
WuJ., ChenJ., WangL., and WangS. (2017). Genome-wide investigation of WRKY transcription factors involved in terminal drought stress response in common bean. Front Plant Sci 8,380.
62.
WuK.L., GuoZ.J., WangH.H., and LiJ. (2005). The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res, 12, 9–26.
63.
XieT., ChenC., LiC., LiuJ., LiuC., and HeY. (2018). Genome-wide investigation of WRKY gene family in pineapple: evolution and expression profiles during development and stress. BMC Genomics 19,490.
64.
YangQ.C., and SunY. (2011). The history, current situation and development of alfalfa breeding in China. Chin J Grassl, 33, 95–101.
65.
YangQ.C., KangJ.M., ZhangT.J., LiuF.Q., LongR.C., and SunY. (2016). Distribution, breeding and utilization of alfalfa germplasm resources. Chin Sci Bull, 61, 261–270.
66.
ZentellaR., ZhangZ.L., ParkM., ThomasS.G., EndoA., MuraseK., et al. (2007). Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis. The Plant Cell, 19, 3037–3057.
67.
ZhangL., ChenL., and YuD. (2018). Transcription factor WRKY75 interacts with DELLA proteins to affect flowering. Plant Physiol, 176, 790–803.
68.
ZhangY., and WangL. (2005). The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol, 5, 1–12.
69.
ZhengJ., LiuF., ZhuC., LiX., DaiX., YangB., et al. (2019). Identification, expression, alternative splicing and functional analysis of pepper WRKY gene family in response to biotic and abiotic stresses. PLoS One 14,e0219775.
70.
ZhouQ., LuoD., ChaiX., WuY., WangY., NanZ., et al. (2018). Multiple regulatory networks are activated during cold stress in Medicago sativa L. Int J Mol Sci 19,3169.
71.
ZhouQ.Y., TianA.G., ZouH.F., XieZ.M., LeiG., HuangJ., et al. (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.
72.
ZouX., NeumanD., and ShenQ.J. (2008). Interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in aleurone cells. Plant Physiol, 148, 176–186.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
