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. 2024 Mar 6;14:5522. doi: 10.1038/s41598-024-55849-1

Genome-wide identification of WRKY transcription factor family members in Miscanthus sinensis (Miscanthus sinensis Anderss)

Yongkang Yan 1,✉, Zhanyou Yan 2, Guofang Zhao 3
PMCID: PMC10918066  PMID: 38448638

Abstract

Miscanthus is an emerging sustainable bioenergy crop whose growing environment is subject to many abiotic and biological stresses. WRKY transcription factors play an important role in stress response and growth of biotic and abiotic. To clarify the distribution and expression of the WRKY genes in Miscanthus, it is necessary to classify and phylogenetically analyze the WRKY genes in Miscanthus. The v7.1 genome assembly of Miscanthus was analyzed by constructing an evolutionary tree. In Miscanthus, there are 179 WRKY genes were identified. The 179 MsWRKYs were classified into three groups with conserved gene structure and motif composition. The tissue expression profile of the WRKY genes showed that MsWRKY genes played an essential role in all growth stages of plants. At the early stage of plant development, the MsWRKY gene is mainly expressed in the rhizome of plants. In the middle stage, it is mainly expressed in the leaf. At the end stage, mainly in the stem. According to the results, it showed significant differences in the expression of the MsWRKY in different stages of Miscanthus sinensis. The results of the study contribute to a better understanding of the role of the MsWRKY gene in the growth and development of Miscanthus.

Keywords: Miscanthus sinensis, WRKY, Phylogenetic analysis, Biotic stress, Expression profiling

Subject terms: Molecular biology, Plant sciences

Introduction

WRKY transcription factors (TFs) were widely distributed in plants, which were first discovered in sweet potato (Ipomoea batatas)1. With genome-wide analyses of different species, WRKY genes have been identified in more species, that included 66 WRKY genes in Arabidopsis2, 119 WRKY genes in maize3, 94 WRKY genes in sorghum4, 79 WRKY genes in potato5, 70 WRKY genes in chickpea6, and 61 WRKY genes in cucumber7. The WRKY protein contains a conserved WRKYGQK motif at its N-terminal and a 60-amino acid-long zinc finger motif at its C-terminus8. Zinc finger motifs can be classified as C2H2 or C2HC. WRKY proteins can be classified into three categories (I, II, III) based on the number of WRKY domains and the type of zinc finger motif9. Group I members have two WRKY domains and zinc fingers of type C2H2. Group II members have only one WRKY domain and one C2H2 zinc finger motif, and Group III members have one WRKY domain and one C2HC zinc finger. Group II can be further subdivided into five subgroups: IIa, IIb, IIc, IId, and IIe10,11.

Studies have shown that WRKY TFs play an important regulatory role in plant growth and development12. For example, AtWRKY12 and AtWRKY13 can exert important regulation on the flowering process of Arabidopsis13. The over-expression of GsWRKY20 from Glycine soja in Arabidopsis also altered the plant's flowering process14. However, OsWRKY11 in rice can control flowering time and plant height15. These depend on WRKY genes being expressed at different times in different parts of the plant. Similarly, the expression of these genes also affects various aspects of plants, such as hormone regulation and stress resistance16.

For example, overexpression of OsWRKY45 in rice enhances disease resistance and drought resistance17. The AtWRKY25 and AtWRKY33 in Arabidopsis thaliana enhance salt tolerance18. Some AcWRKY TFs of kiwifruit were up-regulated under salt stress19. Considering the important role of WRKY TFs in plant growth and development, the study of these genes is very important for agricultural production. The overexpression of TaWRKY2 in wheat enhanced the tolerance to drought stress and increased the yield20. Vitis amurensis VaWRKY12 gene enhanced the cold tolerance of transgenic grape callus21.

Most studies have focused on the genes of annual grasses, but few studies have focused on the genes of perennial grasses. Miscanthus is a perennial grass that has historically been cultivated as a papermaking material and as an ornamental plant. In recent years, Miscanthus has played a role in the direction of ecological restoration and sustainable bioenergy crops22. Therefore, the study has great significance for the genetic improvement of Miscanthus. The improvements can increase productivity and ensure that the crops remain robust despite persistent biological and abiotic stresses. As an essential stress resistance gene, WRKY TFs have high research value. The availability of the whole genome assembly of Miscanthus sinensis enabled identifying MsWRKY. The identification and study of MsWRKY are helpful in understanding the mechanism of plant stress resistance. The genomes of at least four Miscanthus species (M. sacchariflorus, M. sinensis, M. lutarioriparius, and M. floridulus) have been sequenced. M. sinensis was used as the object of this study. M. sacchariflorus has strong stress resistance and large biomass. However, the growth cycle of the plant is too long for commercial biomass production. M. lutarioriparius and M. floridulus have a narrow distribution range and are only found in parts of East Asia. Their biomass yield is low, and the fiber content is too high. Therefore, they are not suitable for the bioenergy development. The function of the Miscanthus WRKY family has been identified and characterized by various methods.

Material and methods

Identification of WRKY family genes in Miscanthus

The genomic data for Miscanthus were obtained from Phytozome 13 (https://phytozome-next.jgi.doe.gov/). From these data, the putative MsWRKY genes can be identified. The database contains the amino acid sequences of Miscanthus WRKY proteins. The Joint Genome Institute (JGI) (https://phytozome.jgi.doe.gov/pz/portal.html#) and the WRKY domain ID were used to identify the potential WRKY (PF03106) proteins of Miscanthus. The data used was Miscanthus sinensis v7.1. To ensure the quality of the data, the CD-HIT suite (https://github.com/weizhongli/cdhit) and Simple Modular Architecture the Research Tool (SMART) (http://smart.embl-heidelberg.de/#) were used to process the resulting sequence23. The duplicate sequences and incomplete sequences were removed.

At the same time, the ExPASy proteomic server (http://web.expasy.org/protparam) was used to predict the physical and chemical properties of the proteins. Their isoelectric point (pI) and molecular weight (MW) were obtained.

Chromosome mapping and classification and phylogenetic analysis of MsWRKY genes

The chromosomal locations of all identified MsWRKY genes were obtained from the Phytozome BioMart tool (https://phytozome.jgi.doe.gov/biomart/martview/). And the MG2C v2.1 (mg2c.iask.in/mg2c_v2.1/) was used for Chromosome mapping of MsWRKY genes.

In constructing a phylogenetic tree to classify MsWRKY genes, we need to use Sorghum bicolor WRKY amino acid sequences. The data were obtained from The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/). They will be used with our MsWRKY sequences. The MEGA v7.0 (https://www.megasoftware.net/) for constructing a phylogenetic tree used multiple sequence alignments with ClustalW to process SbWRKY and MsWRKY protein sequences. The Neighbor-Joining method and the p-distance model were used in the process, and the pairwise deletion and 1000 bootstrap replicates were selected24. Eventually, the phylogenetic tree of SbWRKY and MsWRKY sequences was obtained. Thus, the unknown MsWRKY genes can be divided into different groups and subgroups. By using the sequence alignment data and the phylogenetic tree, the putative Miscanthus WRKY orthologs in Arabidopsis can be identified5.

Gene structure analysis and conserved motif distribution analysis of MsWRKY genes

The genomic sequence and coding sequence (CDS) of each MsWRKY gene can be used to predict the gene structure of the MsWRKY gene. The exon–intron structure of MsWRKYs was analyzed using TBtools25.

The Multiple Em for Motif Elicitation (MEME) (v.5.5.3; https://meme-suite.org/meme/tools/meme) used the parameters which are: maximum motif number: 20; site distributions: any number of repetitions; minimum and maximum width: 6 and 50, respectively to distinguish MsWRKY proteins with conserved motif.26.

Gene ontology annotation and analysis of cis-acting elements of MsWRKY genes

For the gene ontology (GO) annotation analysis of the obtained MsWRKY proteins, the eggNOG-mapper 2.1.12 (http://eggnog-mapper.embl.de/)27 was used. Then, the TBtools was used to map and annotate the obtained data. Ultimately, the biological processes, molecular functions and cellular components of these proteins were obtained.

The online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) analyzed 2000 bp of the upstream region for all MsWRKY genes to analyze the cis-acting elements of MsWRKY genes. It provided the cis-acting elements of MsWRKY genes.

Synteny analysis of MsWRKY genes

The Multiple Collinearity Scan toolkit (MCScanX) was used to examine the gene duplication events with the default parameters. The TBtools was the platform used to analyze the data. The Evalue of the blastP was 15. To explore the syntenic relationships of the WRKY genes obtained from Miscanthus and other selected species, syntenic analysis maps were constructed using MCScanX.

Digital expression pattern analysis of MsWRKY genes

The TBtools analyzed the Miscanthus transcriptomic array data to make heatmaps of the MsWRKY expression profiles to survey MsWRKY expression profiles. In the meantime, the Miscanthus transcriptomic array data was obtained from the JGI database (https://phytozome-next.jgi.doe.gov/).

Results

Identification of WRKY family members in Miscanthus

To identify members of the MsWRKY family, the WRKY domain consensus sequence (PF03106) and the keyword WRKY were used to search in the database. The most complete genome assembly for Miscanthus (Miscanthus sinensis v7.1) in the JGI database was selected. In the MsWRKY family, a conserved domain exists as the basic criterion for inclusion of genes. Using the conserved domain called the WRKYGQK or WRKYGQK-like conserved domain, 203 genes were identified in the JGI database. In these genes, the duplicates and incomplete were removed by the multiple sequence alignments of MEGA 7.0. At the same time, we removed the severely incomplete gene, identified the location of WRKY domain, and kept some of the incomplete genes because they clearly belonged to the MsWRKY family. It was done using the SMART database. In the end, there are a total of 179 non-redundant MsWRKY sequences. The sequences and proteins of these genes are summarized in Table 1.

Table 1.

Characteristics of the identified MsWRKY genes.

Gene name Gene locus ID Chromosome location Gene start Gene end pI MW Conserved heptapeptide Zinc finger type Domain number Group Protein length (aa)
MsWRKY01 Misin01G001000.1 Chr01 256,399 260,899 5.96 42,019.13 WRKYGQK C2H2 1 IIe 388
MsWRKY02 Misin01G047900.1 Chr01 7,447,384 7,449,297 9.28 45,084.5 WRKYGQK C2H2 1 IId 423
MsWRKY03 Misin01G063100.1 Chr01 9,967,017 9,972,013 6.41 34,538.04 WRKYGQK C2H2 1 IIc 331
MsWRKY04 Misin01G064300.1 Chr01 10,185,437 10,187,725 6.27 44,986.89 WRKYGQK C2H2 2 I 420
MsWRKY05 Misin01G080100.1 Chr01 13,181,193 13,184,725 10.03 43,273.5 WRKYGQK C2H2 1 IId 402
MsWRKY06 Misin01G144700.1 Chr01 27,617,080 27,617,952 9.51 11,896.32 WRKYGQK C2H2 1 IId 108
MsWRKY07 Misin01G144800.1 Chr01 27,658,926 27,661,691 8.79 14,941.48 WKKYGQK C2H2 1 IId 138
MsWRKY08 Misin01G263700.1 Chr01 84,350,262 84,352,266 6.35 47,584.95 WRKYGQK C2H2 1 IIe 441
MsWRKY09 Misin01G315300.1 Chr01 105,230,385 105,233,381 6.67 29,695.24 WRKYGQK C2H2 2 I 270
MsWRKY10 Misin01G341600.1 Chr01 112,898,915 112,899,930 10.23 18,701.01 WRKYGQK C2 1 IId 179
MsWRKY11 Misin01G362700.1 Chr01 118,028,855 118,030,800 5.97 33,081.46 WRKYGQK C2HC 1 III 319
MsWRKY12 Misin01G370500.1 Chr01 119,779,719 119,781,349 8.71 23,777.31 WRKYGEK C2HC 1 III 219
MsWRKY13 Misin02G032200.1 Chr02 4,903,340 4,905,683 9.42 46,713.58 WRKYGQK C2H2 1 IId 439
MsWRKY14 Misin02G061600.1 Chr02 10,345,970 10,348,174 6.33 45,153.2 WRKYGQK C2H2 2 I 419
MsWRKY15 Misin02G062400.1 Chr02 10,531,789 10,536,585 8.28 36,115.94 WRKYGQK C2H2 1 IIc 342
MsWRKY16 Misin02G074100.1 Chr02 12,502,555 12,505,918 10.03 43,237.35 WRKYGQK C2H2 1 IId 403
MsWRKY17 Misin02G115600.1 Chr02 21,231,516 21,233,686 8.81 15,261.74 WKKYGQK C2H2 1 IId 142
MsWRKY18 Misin02G115700.1 Chr02 21,277,852 21,278,769 9.3 17,306.11 WRKYGQK C2H2 1 IId 161
MsWRKY19 Misin02G129800.1 Chr02 24,888,414 24,891,276 5.13 29,550.9 WRKYGQK C2HC 1 III 275
MsWRKY20 Misin02G139500.1 Chr02 27,977,090 27,980,493 9.78 37,940.88 WRKYGQK C2H2 1 IId 351
MsWRKY21 Misin02G258000.1 Chr02 82,777,287 82,779,248 6.21 51,394.67 WRKYGQK C2H2 1 IIe 477
MsWRKY22 Misin02G303500.1 Chr02 98,992,567 99,008,454 7.04 51,566.58 WRKYGQK C2H2 2 I 487
MsWRKY23 Misin02G345000.1 Chr02 109,902,400 109,904,305 5.39 32,934.03 WRKYGQK C2HC 1 III 316
MsWRKY24 Misin02G360900.1 Chr02 113,665,512 113,666,881 8.5 23,918.39 WRKYGEK C2HC 1 III 221
MsWRKY25 Misin03G030100.1 Chr03 7,315,277 7,319,424 5.91 40,003.58 WRKYGQK C2H2 1 IIc 385
MsWRKY26 Misin03G090400.1 Chr03 26,714,328 26,724,781 6.53 65,823.66 WKIYHEK C2H2 1 III 577
MsWRKY27 Misin03G145800.1 Chr03 58,356,338 58,358,329 5.9 34,210.05 WRKYGQK C2HC 1 III 323
MsWRKY28 Misin03G145900.1 Chr03 58,463,020 58,464,851 5.15 32,889.18 WRKYGQK C2HC 1 III 308
MsWRKY29 Misin03G167600.1 Chr03 67,670,898 67,672,930 7.5 34,390.44 WRKYGQK C2H2 1 IIa 322
MsWRKY30 Misin03G167700.1 Chr03 67,713,031 67,714,003 5.67 27,360.52 WRKYGQK C2H2 1 IIa 255
MsWRKY31 Misin03G309200.1 Chr03 99,305,845 99,312,404 6.22 65,765.89 WRKYGQK C2H2 2 I 612
MsWRKY32 Misin03G348200.1 Chr03 105,876,921 105,879,425 6.52 34,512.02 WRKYGQK C2HC 1 III 334
MsWRKY33 Misin04G012600.1 Chr04 2,811,844 2,816,422 5.71 40,011.36 WRKYGQK C2H2 1 IIc 388
MsWRKY34 Misin04G103000.1 Chr04 28,871,792 28,881,760 6.64 180,043.9 WEKFGEK C2H2 1 III 1620
MsWRKY35 Misin04G121800.1 Chr04 37,165,681 37,167,742 8.1 32,376.41 WRKYGQK C2HC 1 III 310
MsWRKY36 Misin04G159500.1 Chr04 59,513,396 59,515,552 5.8 33,477.21 WRKYGQK C2HC 1 III 314
MsWRKY37 Misin04G159600.1 Chr04 59,590,103 59,591,840 6.27 35,403.26 WRKYGQK C2HC 1 III 333
MsWRKY38 Misin04G159700.1 Chr04 59,628,053 59,630,374 6.41 32,262.04 WRKYGQK C2HC 1 III 300
MsWRKY39 Misin04G189100.1 Chr04 70,520,338 70,521,730 8.61 24,998.4 WRKYGQK C2H2 1 IIa 232
MsWRKY40 Misin04G189300.1 Chr04 70,613,566 70,615,037 6.45 29,149.56 WSKYGQK C2H2 1 IIa 272
MsWRKY41 Misin04G224400.1 Chr04 80,781,032 80,788,531 6.07 62,448.14 WRKYGQK C2H2 2 I 584
MsWRKY42 Misin04G335900.1 Chr04 103,115,716 103,123,318 6.2 65,841.98 WRKYGQK C2H2 2 I 613
MsWRKY43 Misin04G395900.1 Chr04 113,184,557 113,186,904 9.49 28,237.28 WRKYGQK C2HC 1 III 266
MsWRKY44 Misin05G004000.1 Chr05 1,062,340 1,065,068 6.95 40,831.11 –––- C2H2 0 IIb 398
MsWRKY45 Misin05G004600.1 Chr05 1,127,680 1,130,708 9.02 61,031.03 WRKYGQK C2H2 1 IIb 586
MsWRKY46 Misin05G039300.1 Chr05 8,714,579 8,716,081 6.51 25,862.72 WRKYGKK C2H2 1 IIc 247
MsWRKY47 Misin05G039400.1 Chr05 8,724,881 8,727,718 6.74 54,286.43 WRKYGQK C2H2 1 IIb 529
MsWRKY48 Misin05G043000.1 Chr05 9,436,264 9,443,373 9.66 29,836.26 WRKYGQK C2H2 1 IIc 282
MsWRKY49 Misin05G044100.1 Chr05 9,661,358 9,662,378 10.01 25,167.29 WRKYGQK –– 1 IIc 243
MsWRKY50 Misin05G133400.1 Chr05 33,763,626 33,768,855 6.92 58,669.54 WRKYGQK C2H2 1 IIb 565
MsWRKY51 Misin05G179500.1 Chr05 61,537,333 61,538,346 5.02 23,472.75 WRK–– C2H2 1 IIc 218
MsWRKY52 Misin05G181000.1 Chr05 62,453,032 62,454,436 9.45 34,432.88 WRKYGQK C2H2 1 IIb 334
MsWRKY53 Misin05G204000.1 Chr05 71,047,834 71,051,020 6.39 38,748.61 WRKYGQK C2H2 1 IIc 358
MsWRKY54 Misin05G204800.1 Chr05 71,261,560 71,263,661 8.16 42,492.78 WRKYGQK C2H2 1 IIe 399
MsWRKY55 Misin05G223600.1 Chr05 76,270,790 76,274,606 7.75 40,925.8 WRKYGQK C2H2 1 IIc 392
MsWRKY56 Misin05G245300.1 Chr05 82,376,407 82,381,492 6.65 27,467.87 WRKYGQK C2H2 1 IIc 257
MsWRKY57 Misin05G247500.1 Chr05 82,585,497 82,587,483 5.09 31,767.99 WRKYGQK C2H2 1 IId 309
MsWRKY58 Misin05G257300.1 Chr05 84,706,704 84,708,239 8.87 23,279.05 WRKYGKK C2H2 1 IIc 217
MsWRKY59 Misin05G266000.1 Chr05 87,076,200 87,078,166 4.84 33,807.07 WRKYGQK C2H2 1 IIe 311
MsWRKY60 Misin05G314300.1 Chr05 97,084,382 97,087,219 6.07 58,493.75 WRKYGQK C2H2 2 I 547
MsWRKY61 Misin05G318500.1 Chr05 97,853,542 97,855,451 6.13 28,871.07 WRKYGQK C2HC 1 III 273
MsWRKY62 Misin05G318700.1 Chr05 97,894,342 97,897,352 5.55 35,408.38 WRKYGQK C2HC 1 III 321
MsWRKY63 Misin05G318800.1 Chr05 97,904,621 97,907,196 5.88 39,828.44 WRKYGQK C2HC 1 III 365
MsWRKY64 Misin05G318900.1 Chr05 97,921,736 97,926,790 5.56 29,563.32 WRKYGQK C2HC 1 III 263
MsWRKY65 Misin05G319000.1 Chr05 97,947,986 97,954,617 5.15 24,871.51 –––- C2HC 0 III 222
MsWRKY66 Misin05G341800.1 Chr05 102,614,804 102,620,037 6.21 37,871.09 WRKYGQK C2H2 1 IId 360
MsWRKY67 Misin06G033600.1 Chr06 8,287,503 8,288,890 6.88 25,986.1 WRKYGKK C2H2 1 IIc 248
MsWRKY68 Misin06G033700.1 Chr06 8,316,137 8,318,460 6.6 54,945.21 WRKYGQK C2H2 1 IIb 535
MsWRKY69 Misin06G035300.1 Chr06 8,827,872 8,837,074 9.84 30,409.98 WRKYGQK C2H2 1 IIc 288
MsWRKY70 Misin06G118100.1 Chr06 32,022,401 32,028,104 6.68 58,207.58 WRKYGQK C2H2 1 IIb 559
MsWRKY71 Misin06G173600.1 Chr06 64,362,468 64,364,136 5.41 25,009.44 WRKYGKK C2H2 1 IIc 230
MsWRKY72 Misin06G176500.1 Chr06 65,837,433 65,838,824 9.46 34,595.22 WRKYGQK C2H2 1 IIb 330
MsWRKY73 Misin06G194000.1 Chr06 71,485,939 71,489,452 6.64 37,778.45 WRKYGQK C2H2 1 IIe 352
MsWRKY74 Misin06G201700.1 Chr06 74,689,039 74,691,538 6.65 35,420.7 WRKYGQK C2HC 1 III 341
MsWRKY75 Misin06G223300.1 Chr06 81,494,458 81,497,861 6.95 40,981.74 WRKYGQK C2H2 1 IIc 394
MsWRKY76 Misin06G257000.1 Chr06 89,646,615 89,650,205 6.29 27,463.97 WRKYGQK C2H2 1 IIc 256
MsWRKY77 Misin06G263000.1 Chr06 91,159,450 91,161,480 4.82 33,231.57 WRKYGQK C2H2 1 IIe 308
MsWRKY78 Misin06G304500.1 Chr06 98,736,612 98,740,191 5.98 26,953.02 WRKYGQK C2HC 1 III 238
MsWRKY79 Misin06G304600.1 Chr06 98,786,343 98,796,970 5.61 29,373.08 WRKYGQK C2HC 1 III 262
MsWRKY80 Misin06G304700.1 Chr06 98,801,362 98,804,326 7.08 39,644.48 WRKYGQK C2HC 1 III 365
MsWRKY81 Misin06G304800.1 Chr06 98,822,340 98,825,116 6.14 33,304.99 WRKYGQK C2HC 1 III 308
MsWRKY82 Misin06G304900.1 Chr06 98,833,287 98,834,794 5.98 29,436.72 WRKYGQK C2HC 1 III 276
MsWRKY83 Misin06G308400.1 Chr06 99,650,347 99,653,285 6.65 56,227.46 WRKYGQK C2H2 2 I 524
MsWRKY84 Misin06G319700.1 Chr06 101,544,841 101,551,366 7.72 35,540.77 WRKYGQK C2H2 1 IId 337
MsWRKY85 Misin07G063600.1 Chr07 11,867,503 11,869,447 8.81 38,782.96 WRKYGQK C2H2 1 IIa 361
MsWRKY86 Misin07G113800.1 Chr07 22,968,339 22,970,872 5.77 40,000.76 WRKYGQK C2H2 1 IIe 375
MsWRKY87 Misin07G139300.1 Chr07 29,364,149 29,365,802 9.23 11,245.87 WRKYGQK C2H2 2 I 98
MsWRKY88 Misin07G156800.1 Chr07 33,883,167 33,890,997 6.45 62,499.9 WRKYGQK C2H2 2 I 577
MsWRKY89 Misin07G221700.1 Chr07 48,363,328 48,367,783 6.18 73,536.66 WRKYGQK C2H2 2 I 680
MsWRKY90 Misin07G320900.1 Chr07 80,414,542 80,415,531 6.49 35,113.11 WRKYGQK C2HC 1 III 329
MsWRKY91 Misin07G336100.1 Chr07 97,182,907 97,186,054 9.53 32,283.89 WRKYGQK C2H2 1 IId 304
MsWRKY92 Misin07G427800.1 Chr07 133,666,880 133,670,859 8.74 26,212.78 WRKYGQK C2H2 1 IIc 237
MsWRKY93 Misin07G453800.1 Chr07 139,859,854 139,863,939 5.97 50,932.84 WRKYGQK C2H2 1 IIe 487
MsWRKY94 Misin07G454800.1 Chr07 140,141,540 140,145,937 5.74 50,614.47 WRKYGQK C2H2 1 IIe 487
MsWRKY95 Misin07G514500.1 Chr07 153,014,613 153,017,365 5.33 60,875.02 WRKYGQK C2H2 1 IIb 575
MsWRKY96 Misin08G064100.1 Chr08 13,397,841 13,399,660 8.81 38,153.22 WRKYGQK C2H2 1 IIa 357
MsWRKY97 Misin08G113400.1 Chr08 31,140,937 31,143,013 5.6 39,155.69 WRKYGQK C2H2 1 IIe 368
MsWRKY98 Misin08G130600.1 Chr08 40,498,773 40,500,342 9.46 32,546.22 WRKYGQK C2H2 1 IId 305
MsWRKY99 Misin08G223600.1 Chr08 74,637,099 74,641,364 8.47 26,138.66 WRKYGQK C2H2 1 IIc 236
MsWRKY100 Misin08G295700.1 Chr08 89,997,647 90,000,423 5.41 64,800.38 WRKYGQK C2H2 1 IIb 604
MsWRKY101 Misin09G094400.1 Chr09 31,825,786 31,831,560 6.09 142,969.7 WRKYGQK C2H2 1 III 1269
MsWRKY102 Misin09G124800.1 Chr09 48,969,562 48,973,277 9.32 23,326.47 WRKYGQK C2H2 1 IIc 212
MsWRKY103 Misin09G168300.1 Chr09 69,478,619 69,480,158 5.16 35,435.27 WRKYGEK C2HC 1 III 315
MsWRKY104 Misin09G168400.1 Chr09 69,516,272 69,517,802 5.23 35,384.37 CRKYGEK C2HC 1 III 315
MsWRKY105 Misin10G075100.1 Chr10 22,664,223 22,667,110 7.58 68,606.04 WRKYGR- C2HC 1 III 602
MsWRKY106 Misin10G075200.1 Chr10 22,667,111 22,669,574 11.79 21,954.27 –––- C2 0 III 197
MsWRKY107 Misin10G090500.1 Chr10 28,806,465 28,809,996 9.46 23,366.54 WRKYGQK C2H2 1 IIc 211
MsWRKY108 Misin10G147700.1 Chr10 57,752,929 57,755,316 9.09 45,494.27 WNKYSQK C2HC 1 III 402
MsWRKY109 Misin10G168900.1 Chr10 64,290,186 64,291,933 8.31 28,207.55 WRKYGEK C2HC 1 III 251
MsWRKY110 Misin11G033500.1 Chr11 21,369,493 21,371,062 9.69 31,121.11 WRKYGQK C2H2 1 IId 290
MsWRKY111 Misin11G109800.1 Chr11 50,960,040 50,966,140 6.99 76,641.46 WRKYGQK C2H2 2 I 722
MsWRKY112 Misin11G161900.1 Chr11 61,907,380 61,911,291 8.87 26,425.98 WRKYGQK C2H2 1 IIc 241
MsWRKY113 Misin11G172000.1 Chr11 63,970,651 63,972,274 10.11 32,836.5 WRKYGQK C2H2 1 IId 311
MsWRKY114 Misin11G177600.1 Chr11 64,766,776 64,770,389 5.42 51,673.75 WRKYGQK C2H2 1 IIe 490
MsWRKY115 Misin12G114700.1 Chr12 53,371,560 53,377,084 6.71 76,479.3 WRKYGQK C2H2 2 I 721
MsWRKY116 Misin12G168300.1 Chr12 64,849,948 64,854,472 8.58 26,693.31 WRKYGQK C2H2 1 IIc 246
MsWRKY117 Misin12G221100.1 Chr12 75,273,661 75,277,569 5.4 51,312.26 WRKYGQK C2H2 1 IIe 490
MsWRKY118 Misin12G224200.1 Chr12 75,983,746 75,985,597 10.12 33,577.23 WRKYGQK C2H2 1 IId 320
MsWRKY119 Misin13G053300.1 Chr13 15,284,932 15,287,835 9.19 23,715.16 WRKYGQK C2H2 2 I 214
MsWRKY120 Misin13G053800.1 Chr13 15,379,156 15,382,102 8.1 24,999.43 WRKYGQK C2H2 2 I 225
MsWRKY121 Misin13G065100.1 Chr13 19,010,992 19,011,900 9.97 31,219.41 WRKYGQK C2H2 1 IId 302
MsWRKY122 Misin13G077200.1 Chr13 26,514,539 26,521,340 6.14 61,765.12 WRKYGQK C2H2 2 I 568
MsWRKY123 Misin14G000600.1 Chr14 88,281 90,014 8.39 20,741.62 WRKYGQK C2H2 2 I 187
MsWRKY124 Misin14G044100.1 Chr14 10,368,810 10,372,904 9.05 25,295.99 WRKYGEK C2HC 1 III 225
MsWRKY125 Misin14G044300.1 Chr14 10,410,602 10,412,653 6.45 30,496.32 WRKYGQK C2HC 1 III 269
MsWRKY126 Misin14G044400.1 Chr14 10,415,330 10,420,545 9.23 22,949.89 WRKYGQK C2 1 III 197
MsWRKY127 Misin14G044700.1 Chr14 10,486,916 10,488,031 5.53 30,704.25 WRKYGQK C2HC 1 III 271
MsWRKY128 Misin14G044900.1 Chr14 10,550,794 10,554,423 5.75 32,763.68 WRKYGQK C2HC 1 III 316
MsWRKY129 Misin14G045000.1 Chr14 10,578,622 10,579,241 8.84 15,262.13 WRKYGQK –– 1 III 146
MsWRKY130 Misin14G045100.1 Chr14 10,615,079 10,617,387 5.35 39,100.04 WRKYGQK C2HC 1 III 369
MsWRKY131 Misin14G055800.1 Chr14 13,292,467 13,294,425 5.81 33,108.79 WRKYGQK C2HC 1 III 311
MsWRKY132 Misin14G094800.1 Chr14 36,988,970 36,997,185 7.26 51,967.19 WRKYGQK C2H2 2 I 489
MsWRKY133 Misin14G145800.1 Chr14 53,975,422 53,978,412 10.11 39,116.59 WRKYGQK C2H2 1 IId 367
MsWRKY134 Misin15G021700.1 Chr15 5,345,780 5,348,381 6.01 37,525.59 WRKYGQK C2HC 1 III 354
MsWRKY135 Misin15G022300.1 Chr15 5,508,198 5,510,603 5.95 34,408.46 WRKYGQK C2HC 1 III 301
MsWRKY136 Misin15G022400.1 Chr15 5,519,610 5,521,171 6.41 30,314.18 WRKYGQK C2HC 1 III 269
MsWRKY137 Misin15G022600.1 Chr15 5,554,241 5,557,091 9.24 25,266.01 RRKYGEK C2HC 1 III 224
MsWRKY138 Misin15G022700.1 Chr15 5,568,564 5,569,944 6.24 36,949.21 WRKYGEK C2HC 1 III 349
MsWRKY139 Misin15G059000.1 Chr15 14,509,029 14,511,457 5.86 34,045.56 WRKYGQK C2HC 1 III 321
MsWRKY140 Misin15G117000.1 Chr15 44,938,827 44,944,840 7.26 52,373.63 WRKYGQK C2H2 2 I 494
MsWRKY141 Misin15G165100.1 Chr15 61,759,600 61,762,535 10.06 39,406.88 WRKYGQK C2H2 1 IId 369
MsWRKY142 Misin15G207300.1 Chr15 72,991,988 72,993,980 5.68 33,773.54 WRKYGQK C2HC 1 III 318
MsWRKY143 Misin16G028000.1 Chr16 6,831,166 6,841,544 6.1 42,064.15 WRKYGQK C2H2 1 IIe 390
MsWRKY144 Misin16G048900.1 Chr16 10,992,577 10,999,877 6.07 55,955.5 WRKYGQK C2H2 1 IIb 539
MsWRKY145 Misin16G081700.1 Chr16 22,116,722 22,118,179 8.38 19,270.16 WRKYGKK C2H2 1 IIc 178
MsWRKY146 Misin16G101800.1 Chr16 37,889,291 37,892,295 6.76 44,475.77 WRKYGQK C2HC 1 III 415
MsWRKY147 Misin16G105500.1 Chr16 40,038,943 40,041,203 8.11 52,702.08 WRKYGQK C2H2 2 I 498
MsWRKY148 Misin16G172200.1 Chr16 63,685,681 63,688,656 6.24 59,899.74 WRKYGQK C2H2 2 I 567
MsWRKY149 Misin16G174600.1 Chr16 64,449,520 64,453,297 9.67 16,747.88 WRKYGEK –– 1 III 149
MsWRKY150 Misin16G174700.1 Chr16 64,489,809 64,491,209 4.8 33,581.52 WRKYGQK C2HC 1 III 305
MsWRKY151 Misin16G203800.1 Chr16 69,942,927 69,944,332 9.25 25,824.25 WRKYGQK C2H2 1 IIc 245
MsWRKY152 Misin16G214500.1 Chr16 71,981,919 71,983,740 6.51 22,679.88 WRKYGKK C2H2 1 IIc 220
MsWRKY153 Misin16G230900.1 Chr16 74,460,403 74,463,496 6.14 45,216.03 WRKYGQK C2H2 1 IIc 427
MsWRKY154 Misin16G238000.1 Chr16 75,949,728 75,952,344 6.47 55,813.4 WRKYGQK C2H2 1 IIb 533
MsWRKY155 Misin16G240400.1 Chr16 76,369,804 76,371,283 5.64 31,057.52 WRKYGQK C2HC 1 III 292
MsWRKY156 Misin16G248100.1 Chr16 78,361,013 78,362,627 7.59 40,882.18 WRKYGQK C2H2 1 IIe 383
MsWRKY157 Misin17G038700.1 Chr17 10,631,690 10,636,844 6.08 55,291.53 WRKYGQK C2H2 1 IIb 532
MsWRKY158 Misin17G083500.1 Chr17 25,376,889 25,385,898 6.96 21,980.17 WRKYGKK C2H2 1 IIc 204
MsWRKY159 Misin17G109300.1 Chr17 38,677,661 38,679,975 6.87 53,141.5 WRKYGQK C2H2 2 I 507
MsWRKY160 Misin17G113800.1 Chr17 41,268,683 41,271,638 6.99 44,427.78 WRKYGQK C2HC 1 III 414
MsWRKY161 Misin17G171600.1 Chr17 64,424,660 64,428,034 6.08 21,848.34 WTKYGEK C2HC 1 III 191
MsWRKY162 Misin17G174100.1 Chr17 65,394,933 65,397,741 6.65 60,054.88 WRKYGQK C2H2 2 I 563
MsWRKY163 Misin17G209900.1 Chr17 73,211,242 73,212,749 9.15 24,778.25 WRKYGQK C2H2 1 IIc 234
MsWRKY164 Misin17G216300.1 Chr17 74,457,742 74,459,617 6.2 22,606.83 WRKYGKK C2H2 1 IIc 218
MsWRKY165 Misin17G243200.1 Chr17 79,583,903 79,586,980 6.37 43,647.54 WRKYGQK C2H2 1 IIc 414
MsWRKY166 Misin17G243900.1 Chr17 79,818,715 79,821,649 6.82 61,955.37 WRKYGQK C2H2 1 IIb 589
MsWRKY167 Misin17G248100.1 Chr17 80,589,893 80,591,407 5.8 31,233.78 WRKYGQK C2HC 1 III 294
MsWRKY168 Misin17G257600.1 Chr17 82,409,895 82,413,771 5.06 35,515.91 WRKYGQK C2H2 1 IIc 336
MsWRKY169 Misin17G258100.1 Chr17 82,719,255 82,721,075 7.6 32,985.94 WRKYGQK C2H2 1 IIe 314
MsWRKY170 Misin18G036700.1 Chr18 8,281,538 8,287,821 8.86 67,058.03 WRKYGQK C2H2 1 IIb 638
MsWRKY171 Misin18G048700.1 Chr18 10,206,417 10,210,611 5.82 41,183.59 WRKYGQK C2HC 1 III 385
MsWRKY172 Misin18G150100.1 Chr18 48,766,642 48,768,157 8.32 35,201.51 WRKYGQK C2H2 1 IIe 321
MsWRKY173 Misin18G152000.1 Chr18 50,380,536 50,382,557 5.18 60,818.16 WRKYGEK C2H2 1 IIe 569
MsWRKY174 Misin18G207000.1 Chr18 69,577,536 69,579,342 9.29 44,127.96 WRKYGQK C2H2 1 IIa 411
MsWRKY175 Misin19G033600.1 Chr19 7,916,396 7,933,365 8.04 65,369.63 WRKYGQK C2H2 1 IIb 624
MsWRKY176 Misin19G046600.1 Chr19 10,576,130 10,580,248 6.01 39,605.9 WRKYGQK C2HC 1 III 371
MsWRKY177 Misin19G148800.1 Chr19 49,190,343 49,191,582 7.58 38,894.29 WRKYGQK C2H2 1 IIe 359
MsWRKY178 Misin19G148900.1 Chr19 49,254,920 49,256,188 7.99 40,336.02 WRKYGQK C2H2 1 IIe 377
MsWRKY179 Misin19G200400.1 Chr19 69,378,305 69,380,812 9.55 36,897.55 WRKYGQK C2H2 1 IIa 343
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These MsWRKYs have been named from MsWRKY01 to MsWRKY179 according to their distribution on the chromosome. The starting point was the upper arm of chromosome 1, moving down to the lower arm (Table 1). The properties of this set of MsWRKY proteins were investigated. The MsWRKY proteins ranged from 98 to 1620 amino acids. Their average length was 368 amino acids. Their predicted MW and pI values ranged from 11,246 to 180,044 and 4.8 to 11.79, respectively.

Chromosome mapping and classification and phylogenetic analysis of MsWRKY genes

The locations of the 179 MsWRKY genes were determined by MG2C v2.1 (Fig. 1). MsWRKY genes were distributed on all 19 Miscanthus chromosomes (Chr). Chr01-Chr19 was the chromosome (Chr) number indicating the names and positions of the MsWRKY. Chr 5 had the highest number of MsWRKYs, with 23, representing 12.9% of the total gene family. It was followed by 18 genes on Chr 6, 14 on Chr 16, and 13 on Chr 17. Chromosomes 9, 12, and 13 had only four MsWRKYs each, the fewest.

Figure 1.

Figure 1

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Distribution of 179 MsWRKY genes on Miscanthus chromosomes.

An unrooted phylogenetic tree to study the evolution of MsWRKY family members was constructed by the multiple sequence alignment and neighbor-joining method in MEGA7.0. The data was full-length protein sequences of 40 SbWRKYs and 179 MsWRKYs. SbWRKYs are used as the basis for grouping. They were from Sorghum bicolor (L.) moench, a Poaceae plant that is similar to Miscanthus sinensis.

179 MsWRKYs could be divided into three major groups (I, II, and III) according to the constructed phylogenetic tree (Fig. 2). Of the 24 MsWRKYs in group I, all of them had two WRKYGQK motifs and 23 of them have two C2H2-type zinc finger motifs (C-X3-4-C-X22-23-H-X1-H), corresponding to two complete WRKY domains. Although the protein encoded by MsWRKY09 had only one zinc finger motif, it belonged to group I on the phylogenetic tree.

Figure 2.

Figure 2

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Phylogenetic tree of WRKY members in Miscanthus and Sorghum. All MsWRKYs genes were further divided into subgroups I, II, and III, and group II was further divided into subgroups IIa, IIb, IIc, IId, and IIe.

Group II had a total of 97 protein sequences and was the largest group, accounting for 54.2% of all putative MsWRKYs. Similar reports can also be found in sorghum, cucumber and chickpeas. Most of these proteins had one WRKY domain and the C2H2-type zinc finger motif (C-X4-5-C-X23-H-X1-H). This group was further divided into five subgroups, IIa, IIb, IIc, IId, and IIe, with 8, 16, 32, 21, and 20 members, respectively. Fifty-eight proteins belong to Group III. The proteins in this group had one WRKY domain and the C2HC-type zinc finger motif (C-X7-C-X23-H-X1-C)28. In summary, the classification of MsWRKYs indicated the diversity of these proteins. An extremely wide range of functions could be performed by these proteins.

Gene structure analysis and conserved motif distribution analysis of MsWRKY genes

The exon–intron structures of MsWRKY family members can illustrate the evolution of MsWRKY family members. The introns number of MsWRKY genes ranged from zero to five, while their size varied. The data showed that genes within the same group had certain similarities in the exon–intron distribution patterns. These results suggest that the MsWRKY genes had important structural diversity. It represented the functional diversity among closely related members of MsWRKYs (Fig. 3).

Figure 3.

Figure 3

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Exon–intron structures of MsWRKY genes. Exon–intron structures of MsWRKY genes were obtained after analysed with TBtools for gene structure. Green bars indicate upstream and downstream UTRs, yellow bars indicate coding sequences (CDS), and black lines indicate introns in the gene diagrams.

MEME (version 5.5.3), used to analyze the conserved motifs of all MsWRKY protein sequences, identified 20 distinct conserved motifs. The distribution of 20 conserved motifs identified by MEME in the different groups of MsWRKYs is shown in Fig. 4. Motifs 1 is the WRKY domain. Similar Motif structures can be found between the genes in the same group or subgroup through the results. Motifs 1, 2, 3, and 4 are found in almost all genes. Motifs 15 and 19 were unique to group I. Motifs 9 and 13 were unique to group IIb. Motifs 12 were unique to group IIe. Motifs 10, 16, and 18 were unique to group III. Some of the motifs shared by different groups. Motif 5 was shared by groups I and IIc, and motif 6 and 7 were shared by groups IIa and IIb.

Figure 4.

Figure 4

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Motif analysis of MsWRKYs.

Gene ontology annotation and analysis of cis-acting elements of MsWRKY genes

The Blast2GO analyzed the Gene ontology (GO) annotations of 179 MsWRKY proteins. The MsWRKY target genes can be categorized into three main categories according to different functional groups. The biological processes, molecular functions, and cellular components together make up the Gene ontology (GO) annotations. Through the enrichment analysis, the involvement of MsWRKY in biological processes, molecular functions, and cellular components is in Fig. 5. Most MsWRKYs are involved in regulating cellular processes, biosynthetic processes, and different metabolic processes. Further analysis showed that most MsWRKYs were involved in the plant's stress to external adversity. Many MsWRKYs have been linked to bacterial infections and environmental stress29. The molecular functions of MsWRKYs are mainly a variety of DNA-binding and gene expression regulation. The cellular component of this protein family is mainly organelle and intracellular organelle. Most of the MsWRKY proteins are located in the cell nucleus.

Figure 5.

Figure 5

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Gene ontology analysis of identified MsWRKYs.

Cis-acting elements are essential sequences in regulating gene expression by transcription factors. An online tool PlantCARE was used to analyze all MsWRKY cis-acting elements and extracted 2000 bp promoter regions upstream of all MsWRKY genes. The result shows that every MsWRKY have many cis-acting elements.

Firstly, many transcription-related cis-acting elements can be found including TATA-box, CAAT-box, A-box, HD-Zip, and W-box. The stress-responsive elements formed an important part in the cis-acting elements. This suggests that MsWRKY plays an important role in plants' resistance to external stress. These cis-acting elements include MBS (Anti-drought stress), LTR (anti-low temperature stress), and WUN-motif (wound-responsive elements). In addition, many of the cis-acting elements were regulated by phytohormones. ABA regulates the ABA-responsive element (ABRE). Methyl jasmonate (MeJA) responsive element (TGACG-motif and CGTCA-motif) is regulated by jasmonate phytohormones30. Finally, there are auxin-responsive elements (AuxRR-core and TGA-element), salicylic acid-responsive elements (TCA-element), etc. Finally, there are many light-responsive elements and other regulatory elements. At least one stress-responsive element is on all of the MsWRKY genes and reflects the potential functional variation of the MsWRKY gene.

Synteny analysis of MsWRKY genes

The segmental duplication events occurring in the Miscanthus WRKY family were investigated by conducting a synteny analysis of the MsWRKY genes using BLASTP and MCScanX in TBtools. As shown in Fig. 6, 19 segmental duplication events involving 53 WRKY genes were observed. Tandem duplication events, which were defined by a chromosomal region within 200 kb containing two or more genes, were widely identified for Miscanthus WRKY genes. A very large tandem duplication event was observed in the chromosome 14. These results suggested that some MsWRKYs were possibly generated by segmental duplication events and that the evolution of MsWRKY genes may have been driven, at least in part, by segmental duplication events.

Figure 6.

Figure 6

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Schematic representations for the interchromosomal relationships of MsWRKYs. Blue lines show duplicated WRKY gene pairs in the Miscanthus genome.

The phylogenetic mechanisms of the Miscanthus WRKY family were further explored by constructing comparative syntenic maps of cucumber associated with four representative species, including two dicots (Arabidopsis and cucumber) and two monocots (sorghum and maize) (Fig. 7). 249, 231, 28, and 27 pairs of genes showed syntenic relationships between the other four species: cucumber, Arabidopsis, sorghum and maize, respectively. A total of 249 WRKY collinear gene pairs between Miscanthus and maize were identified, followed by Miscanthus and sorghum (231), Miscanthus and cucumber (28), and Miscanthus and Arabidopsis (27). Both Miscanthus and maize belong to the Poaceae family, and more than 75.4% of the MsWRKY genes showed a syntenic relationship with WRKYs in maize. But some of MsWRKY genes were associated with more than one syntenic gene pair, indicating that WRKY genes in Poaceae family have gone through multiple rounds of duplication events. This may be the reason why monocotyledonous plants have far more WRKY genes than dicotyledonous plants. Importantly, collinear MsWRKY09/11/60/64/83/85/112/179 genes pairs were observed between Miscanthus and all of the other four species, suggesting that these orthologous pairs may have formed before the divergence of dicotyledonous and monocotyledonous plants.

Figure 7.

Figure 7

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Synteny analysis of WRKYs between Miscanthus and other plant species. The collinear blocks are marked by gray lines, while the collinear gene pairs with WRKY genes are highlighted in the red lines. ‘M. sinensis’, ‘A. thaliana’, ‘C. sativus’, ‘S. bicolor’ and ‘Z. mays’ indicate Miscanthus sinensis, Arabidopsis thaliana, Cucumis sativus, Sorghum bicolor, and Zea mays, respectively.

Digital expression analysis of MsWRKY genes at different seasons and in different tissues

The study of the temporal and spatial expression profiles of MsWRKY genes used the microarray data provided by the JGI database and presented the results as heatmaps by TBtools. the microarray datasets used gene expression data for M. sinensis and included a total of 22 samples. The samples were taken from leaf (7), rhizome (9) and stem (6). The samples were collected from plants at different times of the year and reflected the expression of the MsWRKY gene at different stages of plant growth. 175 of the 179 genes showed differential expression in plants. Most MsWRKY genes are highly expressed in rhizome. The expression patterns of MsWRKY in different growth and development stages were also analyzed. Firstly, the MsWRKY gene is first expressed in rhizome in large quantities during plant growth. Then, some MsWRKY genes were heavily expressed in the leaf. Eventually, some MsWRKY genes are over-expression in the stem when plants wither. The results showed that these genes may be involved in stress response at sensitive developmental stages to improve plant tolerance (Fig. 8).

Figure 8.

Figure 8

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Heatmaps of MsWRKY gene expression. MsWRKY expression levels in different tissues and at different seasons.

By analyzing the gene expression heat map and cis-acting elements together, it can be found that genes activated in different periods have different characteristics. In the early stage of plant development, the expression of the MsWRKY gene is mostly controlled by plant hormones and light regulatory elements. In the middle stage of plant development, the MsWRKY gene expressed in leaves is more regulated by infection and injury. At the end of plant development, MsWRKY genes expressed were mostly regulated by ABA and jasmonic acid, and some were stressed by environmental conditions such as drought. This suggests that the MsWRKY gene plays an important role in the growth of the perennial plant Miscanthus sinensis.

Discussion

WRKY transcription factors (TFs) are widely distributed in the plant kingdom and play a crucial role in stress tolerance. The WRKY gene family comprises 66 genes in Arabidopsis, 119 in maize, 94 in sorghum, 79 in potatoes, 70 in chickpeas, and 61 in cucumbers. By analyzing the genomic assembly of Miscanthus sinensis, 179 WRKY genes were identified. As the Miscanthus sinensis is a paleotetraploid31, the amount of the WRKY genes is much higher than that of normal plants, but this has not received much attention in previous studies. The MsWRKY gene is distributed across all 19 chromosomes of Miscanthus, and we have identified MsWRKY92, located on chromosome 7, as a gene that promotes flowering32. Conserved WRKY domains bind to the W-box motif in the promoter of WRKY target genes, which is the most important feature of the WRKY family33,34. A phylogenetic analysis of all the obtained MsWRKY genes has been performed. We performed a phylogenetic analysis of all the obtained MsWRKY genes and classified them into groups I, II, and III based on the number of WRKY domains and the type of zinc finger motif. group II is further subdivided into five subgroups: IIa, IIb, IIc, IId, and IIe. Group I had 24 MsWRKY genes, group II had 97, and group III had 58. In group II, group IIc had the most MsWRKY genes, with 32. The proportions of these genes are similar to those found in other plants35–37.

Most MsWRKY genes have a very conserved WRKYGQK motif. However, other similar sequences have been found in many genes. (MsWRKY07 MsWRKY12 MsWRKY17 MsWRKY24 MsWRKY26 MsWRKY34 MsWRKY40 MsWRKY46 MsWRKY51 MsWRKY58 MsWRKY67 MsWRKY71 MsWRKY103 MsWRKY104 MsWRKY105 MsWRKY108 MsWRKY109 MsWRKY124 MsWRKY137 MsWRKY138 MsWRKY145 MsWRKY149 MsWRKY152 MsWRKY158 MsWRKY161 MsWRKY164 MsWRKY173) Furthermore, there are some genes that are clearly WRKY genes but are missing this sequence (MsWRKY44 MsWRKY65 MsWRKY106). These differences can seriously affect the ability of MsWRKY proteins to bind to W-box elements, which in turn affects the biological function of these proteins. Similar heptapeptide motif variations have been found in other plants, such as sorghum4. In soybeans, for example, two WRKY genes with WRKYGKK motif do not bind to normal W-box elements and do not work33. Therefore, further studies are necessary to confirm the biological function of these WRKY motif aberrant genes.

Studying the exon–intron structure of MsWRKY genes can reveal population-specific patterns. Similar MsWRKY genes on the evolutionary tree tend to have similar exon–intron patterns. The number of introns in the MsWRKY gene ranges from 0 to 5, and some MsWRKY genes do not contain introns, indicating that some WRKY genes have experienced intron loss events38. Intron-free genes have been found in other organisms. There are three main mechanisms by which intron-free genes are produced: reverse transcription (the integration of sequences from RNA into the genome), duplication of existing intron-free genes, and horizontal gene transfer39. Differences in the intron size of MsWRKY genes may result from gene duplication, inversion, and/or fusion events40. In conclusion, the diverse exon–intron structure of MsWRKY genes reflects the evolutionary diversity of the MsWRKY gene family.

Motif structural studies of the MsWRKY gene family reveal both structural conservatism and diversity. Motifs 1, 2, 3, and 4 correspond to WRKY domains and zinc finger domains, and they are found in most MsWRKY genes. Although most motifs' functions are unclear, their distribution also has certain rules. Motifs 15 and 19 were unique to group I. Motifs 9 and 13 were unique to group IIb. Motifs 12 were unique to group IIe. Motifs 10, 16, and 18 were unique to group III. Some of the motifs shared by different groups included motif 5, shared by groups I and IIc, and motifs 6 and 7, shared by groups IIa and IIb. Motif 8 is a nuclear localization signal (NLS), mainly in groups IId, IIe, and III41. In conclusion, motif analysis clearly demonstrates the structural differences of genes in different groups in MsWRKY. These motifs may reflect that these genes participate in specific biological processes and play similar biological functions.

Studying the cis-acting elements of MsWRKY can obtain more information about the gene expression of the MsWRKY gene family. Firstly, many transcription-related cis-acting elements42 including TATA-box, CAAT-box, A-box and HD-Zip are essential for gene expression, and most are involved in constructing transcription complexes. In addition, there is also a batch of cis-acting elements regulated by phytohormones. ABA regulates the ABA-responsive element (ABRE). Methyl jasmonate (MeJA) responsive element (TGACG-motif and CGTCA-motif) is regulated by jasmonate phytohormones. In addition, there are auxin-responsive elements (AuxRR-core and TGA-element), salicylic acid-responsive elements (TCA-element), and so on. A variety of biological and abiotic stresses also regulate these genes. These cis-acting elements include MBS (Anti-drought stress), LTR (anti-low temperature stress), and WUN-motif (wound-responsive elements). At the same time, W-box was also found in the promoter region of many MsWRKY genes. This suggests that there is also mutual regulation between WRKY genes43,44. Studies on cis-acting elements of MsWRKY reflect the diversity of the MsWRKY gene in gene expression regulation.

Existing transcriptome data of Miscanthus sinensis can be used to analyze the expression of MsWRKY gene in different stages of Miscanthus development and expression patterns in different tissues. Future studies should investigate the function of the MsWRKY gene by studying homologous genes in other model plants (such as Arabidopsis) and related plants (other grasses). Future research should focus on the effect of MsWRKY on plant growth and development and against adverse winter environments. Additionally, exploring the role of the MsWRKY gene in plant stress response would be valuable. Our research results provide a foundation for future studies in this field.

Conclusion

In this study, 179 WRKY genes were identified from Miscanthus sinensis. The identification, chromosome mapping, classification, phylogenetic analysis, gene structure analysis, conserved motif distribution analysis, gene ontology annotation, analysis of cis-acting elements, and digital expression pattern analysis have been performed. Through digital expression pattern analysis, the specific expression of the MsWRKY gene in different developmental stages and different parts of plants was found. At the same time, some MsWRKY genes may play an important role in plant stress resistance. In conclusion, this study is conducive to further research on the important functions of the WRKY gene in response to abiotic and biological stresses.

Supplementary Information

Supplementary Information. (55.2MB, zip)

Author contributions

All authors reviewed the manuscript.

Funding

Overseas Scholar Program in the Hebei Province (C20190514), Science and Technology Project of Hebei Province (15457605D, 144576106D), National Natural Science Foundation of China (12072205).

Data availability

All custom scripts used for parsing and analyzing transposable elements, gene families, and gene expression, as described in Supplementary Notes, are available at JGI and NCBI database [https://data.jgi.doe.gov/refine-download/phytozome?organism=Msinensis&expanded=497&_gl=1*13m4cmx*_ga*MTQwODM0NDIwMy4xNjk4MDQwMzEw*_ga_YBLMHYR3C2*MTY5ODA0MDMwOS4xLjEuMTY5ODA0MDgwMS4wLjAuMA..] [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA575573] [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA346689].

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-55849-1.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information. (55.2MB, zip)

Data Availability Statement

All custom scripts used for parsing and analyzing transposable elements, gene families, and gene expression, as described in Supplementary Notes, are available at JGI and NCBI database [https://data.jgi.doe.gov/refine-download/phytozome?organism=Msinensis&expanded=497&_gl=1*13m4cmx*_ga*MTQwODM0NDIwMy4xNjk4MDQwMzEw*_ga_YBLMHYR3C2*MTY5ODA0MDMwOS4xLjEuMTY5ODA0MDgwMS4wLjAuMA..] [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA575573] [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA346689].

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