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Ning P, Liu C, Kang J, Lv J. (2017) Genome-wide analysis of WRKY transcription
factors in wheat (Triticum aestivum L.) and differential expression under
water deficit condition. PeerJ 5:e3232 https://doi.org/10.7717/peerj.3232
Genome-wide analysis of WRKY transcription factors in wheat
(Triticum aestivum L.) and differential expression under water
deficit treatment
Pan Ning
1
2
3
4
1
, Congcong Liu
2
, Jingquan Kang
3
, Jinyin Lv Corresp.
4
College of Science, Northwest Agriculture and Forestry University, Yangling, Shannxi, China
College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
College of Life Science, Northwest Agriculture and Forestry University, Yangling, Shannxi, China
College of Life Science, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
Corresponding Author: Jinyin Lv
Email address: [email protected]
WRKY proteins, comprising one of the largest transcription factor families in plant
kingdom, play crucial roles in the plant development and stress responses. Despite several
studies on WRKYs in wheat (Triticum aestivum L.) were investigated, functional annotation
information about wheat WRKYs was limited. Here, 171 TaWRKY transcription factors (TFs)
were identified from the whole wheat genome and compared with proteins from other 19
species representing nine major plant lineages. A phylogenetic analysis, coupled with gene
structure analysis and motif determination, divided these TaWRKYs into seven subgroups
(Group I, IIa-e, III). Chromosomal location showed that the most TaWRKY genes were
enriched on four chromosomes, especially on chromosome 3B, and 85 (49.7%) genes were
either tandem (5) or segmental duplication (80), which suggested that though tandem
duplication has contributed to the expansion of TaWRKY family, segmental duplication
probably played a more pivotal role. The cis-acting elements analysis revealed putative
functions of WRKYs in wheat during development as well as under numerous biotic and
abiotic stresses. Finally, the expression of TaWRKY genes in flag leaves, glumes and
lemmas under water stress were analyzed, and we found different TaWRKY genes
preferentially express in specific tissue during the grain-filling stage. Our results provide a
more extensive knowledge on TaWRKYs, which helps to complete the information of WRKY
gene family in wheat, and also contribute to screen more candidate genes for further
investigation on function characterization of WRKYs under various stresses.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2809v1 | CC BY 4.0 Open Access | rec: 15 Feb 2017, publ: 15 Feb 2017
1
Genome-wide analysis of WRKY transcription factors in wheat (Triticum
2
aestivum L.) and differential expression under water deficit treatment
3
Pan Ning1, Congcong Liu3, Jingquan Kang2, Jinyin Lv2*
4
1
College of Science, Northwest A&F University, Yangling, Shaanxi, China
5
2
College of Life Science, Northwest A&F University, Yangling, Shaanxi, China
6
3
College of Animal Science and Technology, Huazhong Agricultural University, Wuhan,
7
Hubei, China
8
9
10
Corresponding Author:
Jinyin Lv
11
12
E-mail address: [email protected]
13
1
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2809v1 | CC BY 4.0 Open Access | rec: 15 Feb 2017, publ: 15 Feb 2017
14
ABSTRACT
15
WRKY proteins, comprising one of the largest transcription factor families in plant kingdom,
16
play crucial roles in the plant development and stress responses. Despite several studies on
17
WRKYs in wheat (Triticum aestivum L.) were investigated, functional annotation information
18
about wheat WRKYs was limited. Here, 171 TaWRKY transcription factors (TFs) were
19
identified from the whole wheat genome and compared with proteins from other 19 species
20
representing nine major plant lineages. A phylogenetic analysis, coupled with gene structure
21
analysis and motif determination, divided these TaWRKYs into seven subgroups (Group I,
22
IIa-e, III). Chromosomal location showed that the most TaWRKY genes were enriched on four
23
chromosomes, especially on chromosome 3B, and 85 (49.7%) genes were either tandem (5)
24
or segmental duplication (80), which suggested that though tandem duplication has
25
contributed to the expansion of TaWRKY family, segmental duplication probably played a
26
more pivotal role. The cis-acting elements analysis revealed putative functions of WRKYs in
27
wheat during development as well as under numerous biotic and abiotic stresses. Finally, the
28
expression of TaWRKY genes in flag leaves, glumes and lemmas under water stress were
29
analyzed, and we found different TaWRKY genes preferentially express in specific tissue
30
during the grain-filling stage. Our results provide a more extensive knowledge on TaWRKYs,
31
which helps to complete the information of WRKY gene family in wheat, and also contribute
32
to screen more candidate genes for further investigation on function characterization of
33
WRKYs under various stresses.
34
Keywords: wheat, WRKY, water deficit, expression
35
INTRODUCTION
36
Plants have evolved a wide range of unique strategies to cope with various biotic and abiotic
37
stresses through physical adaption, molecular and cellular changes (Ahuja et al., 2010; Knight
38
& Knight, 2001). Transcription regulation of gene expression in response to developmental
39
and environment changes, mediated by the DNA-binding transcription factors, is one of the
40
most important regulatory mechanisms in plants (Ahuja et al., 2010; Buscaill & Rivas, 2014).
41
WRKYs, one of the largest families of regulators, have been proven to play key roles in
2
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42
numerous stress responses and some development processes(Rushton et al., 2010). Since the
43
first report of WRKY TFs identified in sweet potato(Ishiguro & Nakamura, 1994), WRKY
44
proteins have been found throughout the plant lineage and also in some diplomonads, social
45
amoebae, fungi incertae seais, and amoeboza in succession(Rinerson et al., 2015).
46
WRKY TFs were defined by the presence of one or two highly conserved WRKY
47
domains (WDs) of 60 amino acid residues, including the almost invariant WRKYGQK
48
heptapeptide at the N-terminus, followed by a C2H2 (C-X4-5-C-X22-23- H-X-H) or C2HC
49
(C-X7-C-X23-H-X-C) zinc-finger structure at the C-terminus (Eulgem et al., 2000; Rushton et
50
al., 2010). The WRKY family members are classified into three groups (I, II and III) based on
51
the number of WDs and the features of their zinc-finger-like motif (Eulgem et al., 2000;
52
Rushton et al., 2010). Group I typically contains two WDs including a C2H2 zinc-finger
53
structure, whereas Group II and III are characterized by a single WD, including a C2H2 and
54
C2HC zinc-finger motif, respectively. Group II can be further divided into five subgroups
55
(IIa-IIe) based on phylogenetic analysis of the WDs (Eulgem et al., 2000; Rushton et al.,
56
2010). Members of WRKY family regulate gene expression through exclusively binding to
57
the W-box (TTGACC/T), a cis-element in the promoter region of target genes (Bakshi &
58
Oelmüller 2014; Ulker & Somssich, 2004)
59
Recently, accumulating evidence has demonstrated that WRKY TFs, as important
60
components of plant signaling web, regulate specific transcriptional programs during plant
61
development, as well as in response to a variety of biotic and abiotic stimuli (Ahuja et al.,
62
2010; Bakshi & Oelmüller, 2014; Rushton et al., 2012; Rushton et al., 2010). For example, 61
63
of the PtrWRKY genes in Populus are induced by biotic and abiotic treatments, such as
64
Marssonina bruuea, salicylic acid (SA), methyl jasmonate (MeJA), wounding, cold and
65
salinity (Jiang et al., 2014). In rice, expression of OsWRKY71 gene is induced by cold
66
stress(Kim et al., 2016), while it also encodes a transcriptional repressor of GA signaling in
67
aleurone cells (Zhang et al., 2004). Five transgenic broccoli lines over-expressing BoWRKY6
68
demonstrated significant increased resistance to downy mildew, with resistant levels from
69
low to very high(Jiang et al., 2016). In addition, WRKY TFs were also implicated to
70
modulate plant development, such as seed development and germination (Raineri et al., 2016;
71
Xie et al., 2007; Zhang & Wang, 2011), root growth (Ding et al., 2015; Ranjan & Sawant,
3
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72
2014) , stem elongation (Yu et al., 2012; Zhang et al., 2011), embryogenesis (Jimmy & Babu
73
2015; Lagace & Matton 2004), senescence (Ricachenevsky et al., 2010; Sakuraba et al.,
74
2016), trichome development (Johnson, 2002).
75
Wheat (Triticum aestivum L.), one of the world’s three main cereal with the highest
76
monetary(Keating et al., 2014), is affected by multi-environment stresses, such as salinity,
77
extreme temperature and especially drought, which is a principal constraint to global
78
production of wheat (Mwadzingeni et al., 2016; Wang et al., 2015). However, the mechanism
79
by which wheat responds to abiotic stress has been still poorly understood, which might due
80
to its large genome (approximately 17GB). The identification and functional characterization
81
of the WRKY family in wheat will contribute to clarify the mechanism of stress response.
82
Several studies on wheat WRKY identification have been reported in succession. Forty-three
83
and 92 putative TaWRKYs were early identified from publicly available EST by Niu et al.
84
(2012) and Zhu et al. (2013), respectively. Okay et al. (2014) characterized 160 TaWRKYs
85
and their expression profiling in RNA-Seq libraries. Recently, Zhang et al. (2016) identified
86
116 WRKYs, and 13 of them were characterized as senescence-associated genes. Here, we
87
provide a more extensive knowledge on TaWRKYs based on the whole genome sequence of
88
wheat. A total of 1113 WRKY TFs were identified in 20 plants representing the nine major
89
evolutionary lineages to gain preliminary insight into the evolution of the WRKY family in
90
Plantae. In addition, we identified 171 TaWRKYs from wheat, and detailed analysis,
91
including gene classification, physical and chemical parameters prediction, phylogenetic
92
analysis, chromosomal location, duplication events, conserved motif determination,
93
exon-intron structure and cis-acting element analysis were employed. Finally, gene
94
expression patterns of TaWRKY genes in flag leaf, glume and lemma tissues under water
95
deficit treatment were further determined using qRT-PCR. These results will helps to
96
complete the information of WRKY gene family in wheat, and also contribute to screen more
97
candidate genes for future functional investigation of TaWRKYs under various stresses.
98
Materials AND Methods
99
Database search and identification of WRKYs
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100
The protein sequences of 20 plants from nine different major taxonomic lineages were
101
downloaded from several public databases. All of the amino acid sequences were obtained
102
from the following sources: the Eudicots Arabidopsis thaliana (At), Populus trichocarpa and
103
the Monocots Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Triticum aestivum
104
(Ta, IWGSC1+popseq.31.pep) and Zea mays, and Basal Magnoliophyta Amborella
105
trichopoda and the Bryophyte Physcomitrella patens and the Lycophyte Selaginella
106
moellendorffii and the Chlorophytes Ostreococcus lucimarinus and the Rhodophytes
107
Cyanidioschyzon merolae
108
info/website/ftp/index.html); the Eudicot Cucumis sativus and Chlorophytes Coccomyxa
109
subellipsoidea C-169, Micromonas pusilla CCMP1545 and Volvox carteri
110
Cyanophora paradoxa
111
OrganismDownload.jsf?organism= PhytozomeV9); the Chlrophyte Ostreococcus tauri,
112
Gymnosperms Picea sitchensis and Rhodophyte Galdieria sulphuraria from NCBI
113
(http://www.ncbi.nlm.nih.gov/protein/). The evolutionary relationship of these 20 species
114
were obtained from NCBI (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/
115
wwwcmt.cgi), and visually displayed by phylogenetic tree using FigTree v1.4.3 program
116
(http://tree.bio.ed.ac.uk/software/figtree/).
from the Ensembl Plants (http://plants.ensembl.org/
and Glaucophyte
from Phytozome (http://genome.jgi.doe.gov/pages/dynamic
117
To identify the WRKY TFs in various species, the HMM profile of the WRKY domain
118
(PF03106) downloaded from the Pfam database (http://pfam.xfam.org) (Finn et al., 2016) was
119
applied as a query to search against the local protein database using HMMsearch program
120
(HMMER3.0 software: http://hmmer.janelia.org/) (Finn et al., 2011) with an E-value cutoff of
121
1.0. The sequences obtained were then submitted to the Pfam database to detect the presence
122
of WRKY domains. The protein sequences containing complete or partial WRKY domains,
123
which may be pseudogenes, incomplete assemblies, sequencing errors or mispredictions
124
(Rinerson et al., 2015), were both considered as putative WRKYs. The physical and chemical
125
properties including number of amino acids (NA), molecular weight (MW), theoretical pI
126
(pI) , grand average of hydropathicity (GRAVY), aliphatic index (AI) and instability index (II)
127
of putative TaWRKY proteins were calculated using the online ExPASy-ProtParam tool
128
(http://web.expasy.org/ protparam/).
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Phylogenetic analysis
130
MEGA7.0 program was employed to construct the unrooted phylogenetic tree of identified
131
WRKY protein domains in Triticum aestivum L. and Arabidopsis thaliana L. using the
132
maximum likelihood method(Kumar et al., 2016). The parameters of the constructed trees
133
were: test of phylogeny: bootstrap (1000 replicates), gaps/missing data treatment: partial
134
deletion, model/method LG model, rates among sites: gamma distributed with invariant sites
135
(G). Only the value of bootstrap more than 60 could be displayed on the tree.
136
Chromosomal location of TaWRKY genes
137
To map the locations of WRKY gene transcripts in Triticum aestivum L., MapInspect software
138
(http://www.softsea.com/download/ MapInspect. html) was employed to visualize the
139
chromosomal distribution of deduced TaWRKY genes according to their initial position and
140
length of chromosome. The chromosomal location information of TaWRKYs was obtained
141
from Ensembl Plants website (http://archive.plants.ensembl.org/Triticum_aestivum/Info/
142
Index).
143
To detect the gene duplication, the CDS sequences of WRKY genes in wheat were blasted
144
against each other (e-value<1e-10, identity > 90%) (Song et al., 2014). Tandem duplicated
145
TaWRKY genes were defined as two or more adjacent homologous genes located on a single
146
chromosome, while homologous genes between different chromosomes were defined as
147
segmental duplicated genes (Bi et al., 2016).
148
Characterization of gene structure, conserved motif and putative cis-acting elements
149
The exon-intron structures of TaWRKY genes were obtained by mapping the CDS to DNA
150
sequences using the Gene Structure Display Server2.0 (http://gsds.cbi.pku.edu.cn/) (Hu et al.,
151
2015). CDS (IWGSC1+popseq.31.cds) and genomic sequences (IWGSC1+popseq.31.dna) in
152
Triticum aestivum L. were downloaded from Ensembl Plants website
153
(ftp://ftp.Ensemblgenomes.org/pub/release-31/plants/fasta/triticum_aestivum/).
154
To discover motifs in TaWRKY protein sequences, the online tool MEME 4.11.2
155
(Multiple Expectation Maximization for Motif Elication: http://meme-suite.org/) was utilized
156
to identify the conserved motifs in full length TaWRKYs (Bailey et al., 2009). The optimized
6
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parameters were as follows: distribution of motifs, zero or one occurrence per sequence;
158
maximum number of motifs, 10; minimum sites, 6; maximum width 60.
159
The 1.5kb upstream of the transcription start site (-1) of all identified TaWRKY transcripts
160
was extracted as promoter to predict cis-acting elements using the PlantCARE online
161
(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot M, 2002). Then
162
statistics derived from hits of various cis-acting elements in all TaWRKY transcripts were
163
made, and displayed by diagram.
164
Plant materials, drought treatment and qRT-PCR
165
A hexaploid winter wheat (Triticum aestivum L.) cv. Zhengyin1 (St1472/506) was taken in
166
our pot experiment, which was carried out from October, 2015 to June, 2016 in a greenhouse.
167
Seeds were sown in each plastic pot filled with 7 kg of soil, earth-cumuli-orthic Anthrosols
168
collected in northwest China. An equivalent of 0.447 g (urea)/kg-1 (soil) and 0.2 g (K2HPO3)/
169
kg-1 (soil) were mixed in soil with a net water content of 29.2% at the largest field water
170
capacity. Water control was carried out from anthesis (April 17, 2016). Normal water supply
171
and artificial soil desiccation were implemented with 70-75（control group）and 45-50%
172
( moderate water stress) of the largest field capacity respectively. Spikes and flag leaves of
173
wheat collected at 0, 1, 3, 5, 10, 15, and 25 days after anthesis (DAA), were immediately
174
frozen in liquid nitrogen and then stored at -80℃ for subsequent analysis. Spikes were
175
separated as glume, lemma, grain, palea, and rachis, and only glume and lemma were used in
176
experiment.
177
Total RNA was extracted from wheat tissues using the Trizol reagent (Tiangen, China)
178
following the manufacturer’s instruction, and then digested with RNase-free DNaes I. The
179
quantity and concentration of RNA was evaluated by UV spectrophotometry. The first strand
180
cDNA synthesis was generated using PrimeScriptTM RT Reagent Kit (TaKaRa, China), and
181
the synthesized cDNA products were diluted 1:9 with nuclease-free water to use in qRT-PCR.
182
Primer Primer 5.0 and AllelelID 6.0 (http://www.premierbiosoft.com/index.html) were used
183
to design gene-specific primers (Table S1). Wheat Tublin was taken as the reference gene.
184
185
The qRT-PCR was carried out using based on SYBR GreenSYBR® Premix Ex TaqTM
(TaKaRa, Japan) according to the manufacturer’s instructions with Bio Rad CFX96TM
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real-time PCR detection system (BioRad, USA). Reaction parameters for thermal cycling
187
were: 95℃ for 30 s, followed by 39 cycles of 95℃ for 5 s and 60℃ for 30 s, and at last a
188
melting curve (65-95℃, at increments of 0.5℃) generated to check the amplification. The
189
gene expression levels were calculated with the 2-ΔΔCT methods (Livak & Schmittgen, 2001),
190
and three biological replicates were used.
191
Results
192
Identification of WRKYs in wheat and comparative analysis
193
To comprehensively analyze and identify WRKY TFs in plants, 20 plants representing the
194
nine major evolutionary lineages were chosen for analysis. After searched by HMMER and
195
detected WRKY domains by Pfam database, a total of 1113 WRKY TFs were obtained (Table
196
S2). The evolutionary relationships of various species and the number of WRKY TFs were
197
shown in Fig.1. Most of terrestrial plants, including Monocots, Eudicots and Bryophytes,
198
contained 86 to 171 WRKY proteins, while Picea sitchenis belonging to the Gymnosperm
199
carried only eight WRKY TFs, which could be due to sequencing unfinished. However, six or
200
less WRKY proteins were found in aquatic algaes, of which the number of WRKY TFs in
201
Rhodophytes and Glaucophytes showed zero. In general, the number of WRKY TFs in many
202
higher plants was more than that in lower plants, which suggested that the WRKY TFs may
203
play an important role in the process of plant evolution. The number of WRKY proteins
204
increased as plants evolved, possibly because of genome duplication.
205
In wheat, a total of 174 WRKY proteins were searched using HMM search program.
206
Subsequently, all obtained sequences were verified by Pfam database, which resulted in the
207
identification of 171 WRKY TFs (Table S3). There were 115 TaWRKYs named as
208
TaWRKY1 to TaWRKY115 based on their chromosome location, and other 56 sequences
209
called TaWRKY116 to TaWRKY171 as they were anchored in the scaffolds. Among the 171
210
TaWRKYs, manual inspection showed that some of them are partial sequences, in which
211
WRKY domains or zinc-finger structures were incomplete or even did not exist.
212
The parameters used to describe the TaWRKY proteins were shown in Table S3.
213
Molecular weight, theoretical pI and aliphatic index could not be computed in sequences
214
containing several consecutive undefined amino acids. The lengths of TaWRKY proteins
8
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215
ranged from 44 (TaWRKY121) to 1,482 residues (TaWRKY78), whereas the PI ranged from
216
4.96 (TaWRKY164) to 10.73 (TaWRKY40). This suggested that different TaWRKYs might
217
operate in various microenvironments (Wang et al., 2014). The values of grand average of
218
hydropathicity were all negative, which indicated that TaWRKY proteins were all hydrophilic.
219
Almost all TaWRKYs were defined as unstable proteins, and only 30 TaWRKYs with
220
instability index less than 40 were considered to be stable proteins.
221
Classification and phylogenetic analysis of TaWRKYs
222
To categorize and investigate the evolutionary relationship of the TaWRKY proteins in detail,
223
we constructed an unrooted maximum-likelihood phylogenetic tree with 262 putative WDs in
224
Arabidopsis and wheat (Fig. 2). Based on the classification of AtWRKYs and primary amino
225
acid structure feature of WRKY (Eulgem et al., 2000), TaWRKYs were classified into three
226
major groups (Groups Ⅰ, Ⅱ and Ⅲ). The 30 TaWRKYs possessing two WDs and C2H2-type
227
zinc finger motifs (C-X3-4-C-X22-23- H-X1 -H) were classified into group I. Group II was
228
comprised of 95 sequences, and each protein contained a single WD and C2H2-type zinc
229
finger structure (C-X4-5-C-X23-H-X1-H). We further divided Group II into five subgroups,
230
including Ⅱa, Ⅱb, Ⅱc, Ⅱd and Ⅱe with 11, 7, 50, 17 and 10 members, respectively. Finally, 45
231
TaWRKYs with a single WD were assigned to Group Ⅲ because of their C2HC zinc-finger
232
structure (C-X6-7-C-X23-28-H-X1-C).
233
As shown in Table S3, besides the highly conserved WRKYGQK motifs, we found three
234
variants in TaWRKYs, namely WRKYGKK (10), WRKYGEK (11) and WSKYGQK (1),
235
which were distributed in subgroup Ⅱc, Ⅲ and TaWRKY157, respectively. In addition, two
236
zinc-finger form variants, C-X6-P-X23-H-X-C and C-X6-F-X23-H-X-C were identified in
237
TaWRKY80 and TaWRKY166, respectively. The unique one in all putative TaWRKYs is
238
TaWRKY157, which contained two WDs but with C2HC-type zinc finger structure
239
(C-X7-C-X23--H-X1-C). The “Group I Hypothesis” sees all WRKY genes evolving from
240
Group I C-terminal WRKY domains (Rinerson et al., 2015). Therefore, TaWRKY157 could
241
be taken as an intermediate type of Group I-III member preliminary, though it was classified
242
into Group Ⅲ in phylogenetic tree.
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243
In this study, the Group Ⅱ was found to be the largest group of WRKY TFs family in
244
wheat. The members in Group Ⅱ accounted for approximately 55.6% of all putative
245
TaWRKYs, which was consistent with Musa balbisiana (Goel et al., 2016), pepper(Diao et
246
al., 2016) and soybean(Song et al., 2016a). Subgroups Ⅱa and Ⅱb were separated from one
247
clade, and Ⅱd and Ⅱe clustered to a branch, which is similar to previous study in wheat (Okay
248
et al., 2014; Zhu et al., 2013)
249
Chromosomal location of TaWRKY genes
250
Among the 171 TaWRKY genes, 115 were mapped onto the 21 wheat chromosomes, and the
251
other 56 were anchored in the scaffolds (TaWRKY116-171) (Table S3, Fig. 3). There were
252
relatively more TaWRKY genes distributed in Chromosomes 3B (18, 15.7%), 5B (11, 9.57%),
253
2A (9, 7.8%) and 5D (9, 7.8%). In contrast, chromosome 6B, 7A and 7B contained only one
254
TaWRKY genes (0.870%). In general, most identified TaWRKY genes were observed in the
255
top and bottom sections of chromosomes and only less in central sections. It suggested that
256
the TaWRKY genes were mapped on the all chromosomes with a significantly non-random
257
and uneven distribution. The TaWRKY genes density in each chromosome was ranged from
258
0.004/Mb (7B) to 0.056/Mb (5D) (Fig. S1).
259
Duplication events of WRKY genes have been found universally in a number of plants,
260
such as peanut (Song et al., 2016b), white pear (Huang et al., 2015) and Brassica napus (He
261
et al., 2016). In this study, we identified 79 TaWRKY gene duplication pairs which
262
corresponded to 85 genes (Table S4, Fig.3). It indicated that some of the TaWRKY genes have
263
more than one duplicated gene, which could be due to the multiple rounds of whole genome
264
duplication in wheat. As shown in Fig. 3, two WRKY tandem duplication clusters
265
(TaWRKY59/TaWRKY60, TaWRKY113/TaWRKY114/TaWRKY115) were identified on
266
chromosome 3B and 7D, respectively. In addition, there were 80 genes have undergone
267
segmental duplication, which were paralogs of WRKY genes on different chromosomes (Bi et
268
al., 2016).
269
Gene structure analysis of WRKY genes in wheat
270
The exon-intron distribution was analyzed to further detect structural features of TaWRKY
271
genes. Figure S2 showed that the number of introns in TaWRKY family genes varied from 0
10
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272
to 5, while 0 to 8 in rice (Xie et al., 2005) and 0 to 22 in Musa acuminate (Goel et al., 2016),
273
respectively. It suggested that WRKYs in wheat show lower gene structure diversity. A total of
274
72 (42.11%) TaWRKY genes with two introns accounted for the largest proportion, followed
275
by 44 (25.73%), 23 (13.45%), 15 (8.77%), 14 (8.19%) and 3 (1.75%) genes, possessing 1, 3,
276
4, 0 and 5 introns, respectively. The distribution pattern of introns and exons was
277
group-specific, which is similar to cassava (Wei et al., 2016) and carrot (Li et al., 2016), and
278
TaWRKY gens belonging to the same subfamily shared similar exon-intron structure. For
279
example, TaWRKYs in Group Ⅲ contained 0-5 introns, approximately 91.11% (41/45) of
280
which possessed 1-2 introns.
281
There were two types of introns (V-type and R-type) located in WRKY domain, which
282
were characterized based on their splice site (Bi et al., 2016; Wang et al., 2014; Xie et al.,
283
2005). V-type introns (phase 0) have a splice site before the V (Valine) residue in C2H2 zinc
284
finger structure, and R-type introns (phase 2) on the R (Arginine) residue of the WRKY
285
domain (Bi et al., 2016; Wang et al., 2014; Xie et al., 2005). In our study, all of the TaWRKY
286
genes (17) in Group IIa and IIb only contained V-type introns except TaWRKY165, which had
287
no intron. However, R-type introns were mostly observed in all the other groups (Group I, IIc,
288
IId, IIe and III) (Fig. S2). It indicated that the intron phases were significantly conserved
289
within the same group but remarkably different between groups (Chen, 2014). These results
290
provided an additional evidence to support the phylogenetic groupings and TaWRKYs
291
classification.
292
Motif composition analysis of TaWRKYs
293
The conserved motifs of WRKY proteins in wheat were predicted to explore the similarity
294
and diversity of motif compositions. A total of 10 distinct motifs, named motif 1-10, were
295
detected using MEME online program (Fig. 4). Of these 10 motifs, motif 1 and 4 contained a
296
WRKYGQK sequence, which is basic feature of TaWRKYs. At least one of them contained
297
in almost all deduced TaWRKYs, except several incomplete proteins, such as TaWRKY11,
298
43 and 162. Motif 1 was observed almost in all groups, whereas motif 4 dispersed in Group I
299
mostly.
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300
As displayed schematically in Fig. 4, TaWRKYs within the same group or subgroup
301
shared similar motif compositions. For instance, motif 6 and 10 were unique to Group I,
302
whereas motif 7 is specific to Group Ⅲ. The motif unique to a particular group is likely to be
303
involved in specific biological process in plants. Therefore, it could be speculated that each
304
family or subfamily of WRKY genes might be responsible for the specific biological process
305
(Goel et al., 2016; Lippok, 2003). Furthermore, members in subgroup IIa and IId showed
306
almost the identical motif distribution pattern, indicating functional similarity among them.
307
Interestingly, these two subgroups were also clustered to a branch in phylogenetic tree.
308
Likewise, the same phenomenon was also observed in subgroup IId and IIe. These results
309
further validated the categorization of TaWRKYs and phylogenetic relationships.
310
Variety of cis-acting elements in the promoter regions of wheat WRKY genes
311
It was reported that cis-acting elements in the promoter of are crucial to gene expression,
312
which is an en essential part of its function (Dehais, 1999; Lescot M, 2002). The 1.5kb
313
upstream promoter regions of all TaWRKYs were used to predict cis-acting elements using the
314
online database PlantCARE. Here we found various cis-acting elements in 142 out of 171
315
TaWRKY genes, while the rest of WRKYs could not be detected because of short sequence in
316
their upstream regions (Table S5, Fig.5). A great deal of cis-acting elements were related to
317
response of hormones and biotic stresses, including methyl jasmonate (MeJA), abscisic acid
318
(ABA), salicylic acid (SA), gibberellins(GA), auxin (IAA), zein and fungus.
319
MeJA-responsive elements with largest portion were found in the promoter regions of 109
320
TaWRKY genes. Additionally, some elements involved in various abiotic stresses, such as
321
light, wound, cold, heat, anaerobic induction and drought, were identified in a large number
322
of TaWRKY genes. A total of 44 light-responsive elements were almost distributed in all of
323
the TaWRKYs. There were also some elements observed in genes may regulate expression of
324
different tissues (seed, root, shoot, leaf, phloem/xylem, endosperm and meristem) in wheat
325
development. Interestingly, a total of 76 TaWRKYs contained W-box (TTGACC), which
326
regulates gene expression by binding WRKY, indicating these genes may auto-regulated by
327
itself or cross-regulated with others (Chi et al., 2013; Jiang et al., 2014). MBSI, a MYB
328
binding site involved in flavonoid biosynthetic genes regulation, only existed in TaWRKY87
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329
and TaWRKY142, which suggested that these two genes may regulate flavonoid metabolism.
330
There were two unique genes, TaWRKY58 and TaWRKY94, which might response cold and
331
drought stresses for containing a cold and dehydration responsive elements, C-repeat/DRE.
332
Another special MYB binding site MBS, participated in drought response, were identified in
333
103 genes, indicating that most of TaWRKYs seem to be involved in drought stress response
334
(Table S5). It was noteworthy that all members analyzed contained more than one cis-element.
335
Our analysis and previous studies both suggested that TaWRKY genes are involved in
336
transcriptional regulation of plant growth and stress responses (Bakshi & Oelmüller 2014;
337
Ding et al., 2015; Raineri et al., 2016; Rushton et al., 2010).
338
Expression profiles of TaWRKY genes under drought stress.
339
With the exception of two shorter sequences (TaWRKY122 and 169), 12 out of 171 transcripts
340
were selected as candidate drought responsive genes according to their orthologous WRKYs in
341
Arabidopsis, which are involved in water deprivation, using the Biomart (http://plants.
342
ensembl.org/index.html) (Table S6). The AtWRKYs responding to water stress were obtained
343
based on function annotation in TAIR database (http://www.arabidopsis.org/index.jsp). To
344
validate these candidate 12 drought-response genes, we determined their expression pattern in
345
flag leaves, glumes and lemmas using qRT-PCR. In our study, expression of genes could be
346
detected at the transcript level almost in all selected tissues during the grain-filling period
347
except TaWRKY8 (Table S7, Fig. 6).
348
As shown in Fig. 6, we found that TaWRKY genes in glumes and lemmas share more
349
similar expression pattern compared with in flag leaves. A relatively large group of genes,
350
including TaWRKY1, 20, 31, 112, 123, 142, and 149 were significantly up-regulated in flag
351
leaves at 0, 3 or 5 DAA, which suggested that these genes were highly induced at the early
352
grain-filling stage (0-8 DAA). Among them, some genes, like TaWRKY123 and 142,
353
maintained slightly up-regulated initially and then were restrained followed by an increase in
354
the last point of the drought treatment in glumes. In addition, peaks in the expression of
355
several members (TaWRKY1, 20, 123 and 142) were mostly found at 5DAA in lemma, which
356
lags behind other two tissues. Furthermore, water stress induced the most rapid upregulation
357
of some genes, and showed differences in three tissues. For example, both TaWRKY31 and
13
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TaWRKY149 were induced quickly in flag leaves after the onset of the drought treatment (0
359
DAA), approximately increasing up 6.67 and 8.22-fold, respectively. However, later
360
induction was observed in glumes and lemmas. The immediate transcription response
361
observed upon water stress appeared to be related to a more rapid perception of the drought
362
(Eulgem et al., 2000; Rushton et al., 2010). Genes in another interesting cluster, composed of
363
TaWRKY90, 97, 120 and 133, were down-regulated or slightly changed in glumes and
364
lemmas during the early grain-filling stage, and induced during the middle (9-15 DAA) or
365
late grain-filling stage (16-25 DAA) under drought stress. Whereas, two genes (TaWRKY120,
366
133) of them in flag leaves were strongly induced 3 days after water stress. It indicated that
367
TaWRKY120 and TaWRKY 133 genes were predominantly expressed in flag leaves at the
368
early-filling stage. Our data suggested that the tissues-specific expression of TaWRKYs was
369
existed in wheat, and it appeared to be consistent with their role in tissues.
370
Discussion
371
WRKY TFs are one of the largest families of transcriptional regulators in plants, and form
372
integral parts of signalling webs that regulate many plant process (Rushton et al., 2010).
373
Although some investigation on wheat WRKYs have been reported in sucession,
374
characterization and functional annotation information about TaWRKYs was still sufficient.
375
Niu et al. (2012) identified 43 putative TaWRKYs, named TaWRKY1 to TaWRKY43, which
376
were represented with the same names in another study carried out by Okay et al. (2014). Zhu
377
et al. (2013) identified 92 TaWRKYs from the NCBI dbEST and/or the DFCI gene index, and
378
constructed a phylogeny map. A total of 160 TaWRKYs were characterized in terms of their
379
HMM profiles, conserved domains, distribution among WRKY groups and phylogenetic
380
relationships, and discovered some drought responsive members validated in leaf and root
381
tissues (Okay et al., 2014). Recently, Zhang et al. (2016) identified 116 WRKYs, and 13 of
382
them were characterized as senescence-associated genes.
383
Our present study improves the information of WRKYs in wheat, and provides a more
384
comprehensive knowledge based on the wheat genome. We identified WRKYs in 20 species
385
and characterized 171 wheat WRKYs in terms of gene classification, physical and chemical
386
parameters prediction, phylogenetic analysis, chromosomal location, duplication events,
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387
conserved motif determination, exon-intron structure and cis-acting element analysis, which
388
might help to screen candidate stress-responsive genes in wheat for further study.
389
The whole genome duplication can result in divergence and formation of species over
390
time, accompanied with retention or loss of some duplicated genes (Dehal P, 2005). To
391
understand the evolution of WRKY transcription factors family, we identified a total of 1113
392
WRKY proteins in wheat and other 19 species representing the nine major plant lineages.
393
Interestingly, the number of WRKY TFs in many higher plants was more than that in lower
394
plants, which implied that the WRKY TFs might play significant roles during evolution from
395
simpler unicellular to more complex multicellular forms. Thus, we could preliminary
396
speculate that multiple rounds of duplication during evolution might be the main reason for
397
the different numbers of the WRKY proteins in diverse species.
398
Compared with the species analyzed in this study, the wheat (Triticum aestivum L.)
399
genome contained the highest number of WRKY TFs (171). The expansion of WRKY gene
400
family in wheat might be due to the following reasons. (1) Triticum aestivum L. is an
401
allohexaploid, and originated from two recent hybridizations between three diploid
402
progenitors, donors of the A, B and D subgenomes (Glover et al., 2015). Triticum aestivum L.
403
genome experienced the whole genome duplication events after two hybridizations at
404
approximately 0.8 and 0.4 million years ago (MYA), respectively (Glover et al., 2015), which
405
would produce a large number of paralogs (Conant & Wolfe 2008). (2) Likewise, small-scale
406
gene duplication, including segmental and tandem duplication, might be also significant in
407
the evolution of WRKY gene family in wheat. The origin of new genes during evolution was
408
dependent on gene duplication. Gene duplication allows essential genes to undergo mutations
409
in the duplicated copy, suggesting that similar genes would diverge over the long
410
evolutionary time period, and then improving the expansion and evolution of gene family
411
(Conant & Wolfe 2008; De et al., 2008). The current investigate showed that 85 of 171
412
(49.7%) TaWRKY genes evolved from either tandem duplication or segmental duplication.
413
Interestingly, 80 of WRKY genes were segmentally duplicated and only five were tandemly
414
duplicated, implying that high segmental and low tandem duplications existed in TaWRKY
415
genes, consistent with white pear (Huang et al., 2015), grapevine (Wang et al., 2014) and
416
soybean (Song et al., 2016a). Our results showed that number of duplicated genes was mainly
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417
determined by segmental events, because genes generated from segmental duplication have
418
more chance to be retained due to subfunctionalization or neofunctionalization (Huang et al.,
419
2015; Lynch, 2000; Moore & Purugganan, 2005; Wang et al., 2005). Therefore, it was
420
inferred that though tandem duplication has contributed to the expansion of TaWRKY family,
421
segmental duplication probably played a more pivotal role (Zhu Y, 2014). (3) The expansion
422
of gene families along a specific lineage can be due to chance or the result of natural selection.
423
Adaptive expansion of gene families occurs when natural selection would favor additional
424
duplicated genes (Demuth & Hahn, 2009).
425
Increasing research suggests that the WRKY transcription factors are involved in various
426
biological processes, including plant development, responses to biotic and abiotic stresses
427
(Eulgem & Somssich, 2007; Liu et al., 2015; Luo et al., 2013; Rushton et al., 2010; Zhao et
428
al., 2015). Plant hormones, as essential endogenous signal molecules within the plant, can
429
regulate cellular processes, plant growth and development under severe stress conditions
430
(Grove, 1979; Kermode, 2005; Ryu & Cho, 2015). Many evidence indicated that the
431
expression of WRKY genes was affected after hormone treatment (Jiang et al., 2014; Yang et
432
al., 2009). In wheat, a large amount of cis-acting elements responding to phytohormones,
433
such as MeJA, ABA, SA, GA etc., were detected in TaWRKY genes. It suggested that these
434
WRKY genes might regulate growth and development of wheat by functioning as key factors
435
in regulating specific signalling pathways. In addition, WRKY transcription factors were
436
involved in responses to abiotic stresses. For instance, TaWRKY44 in transgenic tobacco
437
confers multiple abiotic stress tolerances, including drought, salt and osmotic stress (Wang et
438
al., 2015). Twelve GmWRKY genes were differentially expressed under salt stress (Song et al.,
439
2016a). In this work, a large number of TaWRKY genes contained several cis-acting elements
440
associated with abiotic stresses, such as light, wound, cold, heat, anaerobic induction and
441
drought, implying that a number of WRKY genes in wheat participate in various abiotic
442
stresses. In general, the results indicated that most of TaWRKY genes were involved in
443
multiple biotic and abiotic stresses, which was consistent with previous study (Eulgem &
444
Somssich, 2007; Jiang et al., 2014).
445
446
Drought is one of the most significant stresses resulting in reduction of wheat production
(Keating et al., 2014). The grain-filling is mainly sustained by photosynthesis of flag leaves
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447
and spikes under drought treatment, and photosynthesis of spike is less sensitive to drought
448
than that in flag leaves (Jia et al., 2015; Tambussi et al., 2005). However, the investigation on
449
TaWRKY genes was mostly focus on root and leaves, and limited in spikes. In this study, we
450
determined relative expression of TaWRKY genes in flag leaves, glumes and lemmas during
451
the grain-filling period upon water deficit using qRT-PCR. Obvious differences of gene
452
expression pattern between flag leaves and spikes (glumes and lemmas) were observed in
453
wheat. For example, TaWRKY142 were up regulated at 3 DAA in flag leaves, but induced in
454
glumes and lemmas since the imposition of the water stress and maintained upregulation
455
during the early grain-filling stage, which suggested the putative role of TaWRKY142 gene in
456
spike tissues. In addition, the majority of TaWRKYs in a group shared similar expression
457
pattern, but some were not. For instance, TaWRKY1, 120 and 133, the orthologs to AtWRKY3,
458
all belong to Group I. Both TaWRKY120 and TaWRKY133 were up regulated at 3DAA and
459
the middle-late grain filling stage in flag leaves, while induction of TaWRKY1 were only
460
observed at 3DAA. The structure analysis showed that TaWRKY120 and TaWRKY133 have
461
similar exon-intron structure and same motif composition, while TaWRKY1 showed
462
difference with them, which may help to explain why TaWRKYs in a group do not necessarily
463
shared the similar expression pattern. The similar phenomenon was also reported in Salvia
464
miltiorrhiza. Five genes (SmWRKY2, 24, 39, 54 and 55), belonging to Group I, were
465
predominantly expressed in roots, whereas the other Group I members, including SmWRKY42,
466
13 and 60 were mainly expressed in stems, leaves and flowers, respectively (Li et al., 2015).
467
Conclusion
468
In this study, our results provide a more extensive knowledge on WRKYs in wheat. With the
469
whole wheat genome sequence, 171 TaWRKYs were identified, and compared with proteins
470
from other 19 species representing nine major plant lineages to gain preliminary insight into
471
the evolution of the WRKY family in Plantae. A phylogenetic analysis, coupled with gene
472
structure analysis and motif determination, divided these TaWRKYs into seven subgroups
473
(Group I, IIa-e, III). Chromosomal location showed that the most TaWRKY genes were
474
enriched on four chromosomes, especially on chromosome 3B, and 85 (49.7%) genes were
475
either tandem or segmental duplication. The cis-acting elements analysis revealed putative
17
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476
functions of WRKYs in wheat during development as well as under numerous biotic and
477
abiotic stresses. Finally, the differential expression of TaWRKY genes in flag leaves, glumes
478
and lemmas under drought stress were analyzed, and we found different TaWRKY genes
479
preferentially express in specific tissue during the grain-filling stage. Taken together, our
480
results will help to complete the information of WRKY gene family in wheat, and also
481
contribute to screen more candidate genes for further investigation on function
482
characterization of WRKYs under various stresses.
483
ACKNOWLEDGEMENTS
484
We are grateful to Mr. Dangxiao Liu and Mr. Huawei Liu for providing valuable suggestions.
485
ADDITIONAL INFORMATION AND DECLARATIONS
486
Funding
487
This study was supported by the projects of the National Natural Science Foundation of
488
China (grant number 31271624). The funders had no role in study design, data collection and
489
analysis, decision to publish, or preparation of the manuscript.
490
Grant Disclosures
491
The following grant information was disclosed by the authors:
492
National Natural Science Foundation of China: 31271624.
493
Competing financial interests
494
The authors declare no competing financial interests.
495
Author Contributions
496
 Pan Ning conceived and designed the experiments, performed the experiments, analyzed
497
the data, wrote the paper, prepared the figures and/or tables, reviewed drafts of the paper.
498
499
 Congcong Liu conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and /or tables.
500
 Jingquan Kang contributed materials, performed the experiments.
501
 Jinyin Lv conceived and designed the experiments, contributed regents, reviewed drafts of
502
the paper.
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503
Data Availability
504
The following information was supplied regarding data availability:
505
The raw data has been supplied as Supplemental Information.
506
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Figure 1(on next page)
Evolutionary relationship of 20 species among nine lineages within the Plantae.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2809v1 | CC BY 4.0 Open Access | rec: 15 Feb 2017, publ: 15 Feb 2017
Species
Number of WRKYs
Brachypodium distachyon
86
Triticum aestivum
171
Oryza sativa
88
Zea mays
163
Sorghum bicolor
96
Populus trichocarpa
122
Cucumis sativus
88
Arabidopsis thaliana
91
Basal Magnoliophytas
Amborella trichopoda
33
Gymnosperms
Picea sitchensis
Lycophytes
8
Selaginella moellendorffii
35
Bryophytes
Physcomitrella patens
117
Monocots
Eudicots
Chlorophytes
Rhodophytes
Glaucophytes
Ostreococcus lucimarinus
2
Ostreococcus tauri
6
Micromonas pusilla CCMP1545
2
Coccomyxa subellipsoidea C-169
1
Volvox carteri
4
Galdieria sulphuraria
0
Cyanidioschyzon merolae
0
Cyanophora paradoxa
0
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Figure 2(on next page)
Phylogenetic tree of WRKY domains from wheat and Arabidopsis.
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AtWRKY16
AtWRKY16
AtWRKY29
AtWRKY22
AtWRKY27
TaWRKY16
3
AtWRKY35
TaWRKY5
TaWRK 1
Y
3
TaW
TaW RKY100
TaW RKY107
TaW RKY119
R
TaW KY15
AtW RKY10 5
Ta RKY1 3
AtWWRKY5 4
At RKY 5
At WRK 69
Ta WRK Y69
TaWWRKYY65
A
R 54
Ta tWR KY89
AtWWRKKY21
AtW
60
RK Y22
Y
T
T aW RKY 15
T aW RKY 15
A aW RK 80
Ta tWR RKY Y91
A W K 1
T tW R Y3 58
T aW R KY 9
T aW R KY 14
Ta aW RK KY1 7 8
W RK Y1 59
RK Y2 54
Y3 7
7
94
82
65
73
65
60
RKY 8
AtW RKY118
AtW RKY 4
AtWRKY1486
Y
TaWaWRKY156
T RK Y87
K
TaW R Y40
TaWWRKY1145
At RK Y16 7
TaWWRK Y16 13
Ta WRK KY1 170
Ta WR KY 115 1
Ta aWR RKY 16 6
T aW RKY KY3 Y9
R K 7
T
W
Ta tW R KY4 Y6
W
A
At WR RK Y42
At AtW RK
W
At
74
65
5
Y3 1
RK Y 1 9
W RK Y 3 3
Ta tW RK KY7 81
A tW R KY 1
A aW R KY7 3
T aW R Y9 7
T aW RK Y1
T aW RK Y67
T tW RK 11
A aW RKY 74
T tW RKY 151
A tW KY 9
A aWR KY5 46
T tWR KY1 1
A WR KY12 2
Ta WR Y12
Ta WRK Y169
80
Ta WRK Y112
Ta WRK 46
Ta WRKY 57
72
At WRKY 8
Ta RKY5 8
91
TaW RKY7 7
TaW RKY13
TaW RKY70
AtW RKY41
AtW RKY41
71
AtW Y53
AtWRK Y30
AtWRK Y110
82
TaWRK 13
TaWRKY
73
TaWRKY46
TaWRKY126
TaWRKY134
TaWRKY29
AtWRKY55
TaWRKY95
TaWRKY153
TaWRKY24
TaWRKY92
TaWRKY
TaWRK 84
TaWRK Y40
AtWRK Y157
AtW YX
TaW RKY55
95
TaWRRKY101
TaW KY59
93
TaW RKY13
78
TaW RKY60 6
RKY7
TaW
T a RKY 6
96
Ta WRKY 72
94
TaWWRKY665
T a RK 6
68
Ta WRK Y74
Ta WR Y70
98
Ta WR KY10
92 61
TaWWRKKY5
T
R Y4
T aW KY
T aW RKY 14
Ta aWR RKY5 9
9967
T W K 6
T aW RK Y44
A aW RK Y6
A tW R Y1 4
At tW RK KY13 32
A W R Y 5
At tW RK KY6 67
W RK Y 6 7
RK Y 6 4
Y6 3
6
62
T
A aW
A tW R
Ta tW RK KY1
R Y 9
W
Ta AtW RK KY6 45
W
A R RK Y52 8
Ta tWR KY1 Y28
Ta WR KY 30
A WR KY5 48
At tWR KY4 0
AtWWRK KY718
At R Y4
T WR KY4 3
70
Ta aWR KY1 3
TaWWRKYKY212
TaW RKY 102
106
R
K
TaW Y1
TaW RKY305
TaW RKY3 8
TaW RKY454
82
TaW RKY49
RK
TaW Y129
AtWR RKY7
82
TaWR KY50
KY
TaW 139
TaWRKRKY2
Y4
2
TaWRKY6
63
3
TaWRKY1
TaWRKY535
AtWRKY51
AtWRKY8
TaWRKY31
AtWRKY57
TaWRKY97
TaWRKY12
TaWRKY123
TaWRKY90
TaWRKY149
TaWRKY207
AtWRKY5
23
AtWRKY Y6
TaWRKY16
K
TaWRKY117
TaWR RKY17
TaW RKY18
TaW KY116
R
TaW tWRKYX6
A KY16
8
R
TaW RKY1124
TaW RKY1
TaW
69
8
Y3 2
RK Y 6 2 5
W RK Y1 40
At tW RK KY1 143
A aW R KY 43
T W R
7
Ta aW RKY Y14 5
T aW RK Y8 0
T aW RK Y16 2
T aW RK 14
T W KY 4
714
9
Ta aWRRKY5 11
T tW KY 2
A aWR KY6 27
R Y1
T
80
TaWWRKKY49 8
Ta tWR KY12
A WR Y61
Ta WRK
Ta
Ta
T WR
At aWR KY1
Ta TaWWRK KY4 31
W
At RK RKY Y31 7
W
6
Ta AtW RK Y10 8
Ta WR RKY Y72 8
68
Ta WRK KY1 61
WR Y 64
1
At K 0
AtWWRKY1624
Y
A RKY 19
At tWR 19
60
At WRK KY
AtWWRKYY4
TaW RK 3
AtW RKY6Y4
TaW RKY3 9
T a RKY 2
TaWWRKY975
4
R
TaW KY150
TaWR RKY82
AtWRKY141
K
Y
5
TaWR
8
AtWR KY30
TaWRKKY10
Y7
AtWRKY27
TaWRKY2
TaWRKY263
TaWRKY96
AtWRKY33
TaWRKY33
TaWRKY88
TaWRKY39
AtWRKY26
AtWRKY34
TaWRKY133
AtWRKY25
97
TaWRKY145
83
TaWRKYY9
9
TaWRK Y44
AtWRKKY44
92
AtWR RKY1
TaW Y120
K
71
TaWRWRKY8
a
T
11
RKY1
TaW RKY1098
79
TaW RKY1671
TaW RKY1Y20
TaWtWRK Y20
A RK 26
AtW RKY152
AtWRKY Y98
K
TaWaWR KY791
T WR KY4 2
Ta WR KY3 5
73
a
T WR KY2 Y1
Ta WR RK Y1
Ta AtW RK 56
Y
4
W
At RK KY2 13
W
At tWR RKY 138 5
A tW KY Y7 8
A WR RK Y2 36
K
Ta AtWWR RKY
Ta aW
T
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Figure 3(on next page)
Chromosome distribution of TaWRKY genes.
The chromosomal position of each TaWRKY was mapped according to the wheat genome.
The chromosome numbers were shown at the top of each chromosome. Fifty six TaWRKYs on
the scaffold (TaWRKY115-171) could not be anchored onto any specific chromosome. The
location of each WRKY gene was pointed out by a line. The scale is in mega bases (Mb). The
black boxes indicated the tandem duplication genes, and the segmental duplicated genes
were shown with red dots.
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Chr 1A
Chr 1D
Chr 1B
25
TaWRKY1
TaWRKY2
75
TaWRKY3
TaWRKY14
TaWRKY15
TaWRKY16
TaWRKY17
TaWRKY18
Mb
125
150
TaWRKY29
TaWRKY22
TaWRKY30
TaWRKY38
TaWRKY39
TaWRKY40
TaWRKY41
Chr 3A
Chr 3D
Chr 4A
Chr 4B
TaWRKY65
TaWRKY63
TaWRKY42
Chr 3B
TaWRKY69
TaWRKY70
TaWRKY71
TaWRKY64
TaWRKY43
TaWRKY66
TaWRKY67
TaWRKY45
TaWRKY46
TaWRKY72
TaWRKY47
TaWRKY48
TaWRKY44
TaWRKY68
TaWRKY33
TaWRKY4
TaWRKY5
TaWRKY6
TaWRKY7
TaWRKY27
TaWRKY9
TaWRKY10
TaWRKY11
275
TaWRKY34
TaWRKY35
TaWRKY12
TaWRKY13
Chr 4D
0
Chr 5A
Chr 5B
75
Mb
100
125
150
175
Chr 6A
Chr 6B
Chr 6D
Chr 7A
Chr 7B
TaWRKY108
TaWRKY101
25
50
Chr 5D
Chr 7D
TaWRKY73
TaWRKY74
TaWRKY81
TaWRKY75
TaWRKY82
TaWRKY83
TaWRKY76
TaWRKY77
TaWRKY78
TaWRKY79
TaWRKY80
TaWRKY106
TaWRKY94
TaWRKY95
TaWRKY96
TaWRKY111
TaWRKY84
TaWRKY85
TaWRKY86
TaWRKY87
TaWRKY88
TaWRKY110
TaWRKY92
TaWRKY93
TaWRKY107
TaWRKY97
TaWRKY98
TaWRKY99
TaWRKY100
TaWRKY109
TaWRKY102
TaWRKY103
TaWRKY104
TaWRKY105
TaWRKY112
200
225
TaWRKY89
TaWRKY90
TaWRKY91
TaWRKY113
TaWRKY114
TaWRKY115
Group
I Subgroup
IIa Subgroup IIb Subgroup IIc Subgroup
IId Subgroup
Group
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| CC BY 4.0 Open
Access | rec:IIe
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publ: 15 Feb 2017
TaWRKY49
TaWRKY50
TaWRKY51
TaWRKY52
TaWRKY53
TaWRKY54
TaWRKY55
TaWRKY56
TaWRKY57
TaWRKY58
TaWRKY59
TaWRKY60
TaWRKY61
TaWRKY62
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
375
400
425
450
475
500
525
550
575
600
625
650
675
700
725
750
Mb
TaWRKY23
TaWRKY24
TaWRKY25
TaWRKY26
200
250
TaWRKY20
TaWRKY21
Chr 2D
TaWRKY36
TaWRKY37
TaWRKY31
TaWRKY32
175
225
Chr 2B
TaWRKY28
TaWRKY8
50
100
Chr 2A
TaWRKY19
0
Figure 4(on next page)
Schematic diagram of conserved motifs in TaWRKYs.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2809v1 | CC BY 4.0 Open Access | rec: 15 Feb 2017, publ: 15 Feb 2017
(A)
Name
p-value
Name
Motif Location
p-value
Motif Location
IIa
IIb
I
IId
IIe
III
IIc
Motif 2
Motif 10
Motif 7
Motif 6
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Motif 9
Motif 8
Motif 5
Motif 3
Motif 4
Motif 1
(B)
Figure 5(on next page)
The number of TaWRKY genes containing various cis-acting elements.
The cis-acting elements were identified with the online PlantCARE program using the 1.5kb
upstream from the transcription start site of TaWRKY genes. The graph was generated based
on the presence of cis-acting element responsive to specific elicitors/conditions/processes (xaxis) in WRKY gene family members (y-axis).
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0
TATA-box
CAAT-box
A-box
OBP-1 site
ATGCAAAT motif
5UTR Py-rich stretch
AT-rich element
circadian
MSA-like
E2Fa
E2Fb
Skn-1_motif
GCN4_motif
CCGTCC-box
CAT-box
OCT
dOCT
NON-box
RY-element
HD-Zip 1
HD-Zip 2
as1
motif I
AC-I
AC-II
ABRE
motif IIb
CE3
CE1
GARE-motif
P-box
TATC-box
TCA-element
SARE
TGA-element
AuxRR-core
TGA-box
AuxRE
O2-site
ERE
CGTCA-motif
TGACG-motif
TC-rich repeats
ARE
GC-motif
ELI-box3
Box-W1
box E
EIRE
AT-rich sequence
W box
box S
GCC box
WUN-motif
JERE
LTR
C-repeat/DRE
HSE
MBS
CCAAT-box
MBSI
MRE
G-box
Sp1
G-Box
GAG-motif
I-box
GT1-motif
MNF1
ACE
Box I
Box 4
GATA-motif
GA-motif
CATT-motif
AE-box
TCT-motif
TCCC-motif
ATCT-motif
3-AF1 binding site
rbcS-CMA7a LAMPelement
ATC-motif
Box III
box II
TGG-motif
chs-Unit 1 m1
chs-CMA2a
Box II
L-box
GTGGC-motif
CG-motif
C-box
Gap-box
CAG-motif
AAAC-motif
4cl-CMA2b
ATCC-motif
GATT-motif
chs-CMA2c
Pc-CMA2c
chs-CMA1a
chs-CMA2b
LS7
CGT-motif
as-2-box
Number of TaWRKY genes
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
Transcription Cell cycle
Development
Hormone
Abiotic / biotic stress
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Type of cis-acting element
Figure 6(on next page)
The expression pattern of TaWRKY genes in flag leaves, glumes and lemmas during the
grain filling stage under drought stress.
(A) Bar graphs showing the relative expression values of each TaWRKY genes after drought
treatment. Samples were collected 0, 1, 3, 5, 10, 15, and 25 days after anthesis, from which
water control was carried out. The mean ± SE of three biological replicates are presented.
Relative fold changes were obtained by qRT-PCR using the 2-ΔΔCT method. (B) Heat map
showing the expression profile of TaWRKYs in flag leaves, glumes and lemmas under drought
stress. Heat map was generated based on log2 transformed count value from three
replicates of qRT-PCR data using R language. Red and green boxes indicated high and low
expression levels of genes, respectively. Blank indicated the gene was not detected.
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15
10
10
Flag leaf
5
5
Glume
5
0
1
3
5
10
15
25
TaWRKY 90
20
15
10
5
0
4
0
1
3
5
10
15
25
Ta WRKY 120
1
3
5
10
15
25
TaWRKY 97
0
20
15
15
10
10
5
5
0
10
0
1
3
5
10
15
25
TaWRKY 123
0
6
0
1
3
5
10
15
25
10
15
25
10
15
25
TaWRKY 112
0
1
3
5
TaWRKY 133
4
6
2
4
1
8
20
0
8
3
0
0
2
2
0
1
3
5
10
15
25
TaWRKY 142
6
0
15
0
1
3
5
10
15
25
0
0
1
3
5
Days after anthesis / d
TaWRKY 149
10
4
5
2
0
0
1
3
5
10
15
25
0
0
Days after anthesis / d
1
3
5
10
15
25
Color Key and Histogram
35
Relative expression
TaWRKY 31
Lemma
10
25
Relative expression
15
TaWRKY 20
20
0
Relative expression
15
TaWRKY 1
Count
Relative expression
25
Days after anthesis / d
0 15
(A)
−2
(B)
Glume
Flag leaf
0d
1d
3d
5d
10d
15d
25d
0d
1d
3d
5d
−1
0
Row Z−Score
1
2
Lemma
10d
15d
25d
0d
1d
3d
5d
10d
15d
25d
TaWRKY001
TaWRKY020
TaWRKY142
TaWRKY123
TaWRKY031
TaWRKY149
TaWRKY112
TaWRKY097
TaWRKY090
TaWRKY120
TaWRKY133
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2809v1 | CC BY 4.0 Open Access | rec: 15 Feb 2017, publ: 15 Feb 2017