bes/bzr transcription factor tabzr2 positively regulates … · 3 60 61 62 abstract 63 bri1-ems...
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BES/BZR Transcription Factor TaBZR2 Positively 1
Regulates Drought Responses by Activation of TaGST11 2
Xiao-Yu Cuia,d,2, Yuan Gaoa,2, Jun Guob, Tai-Fei Yua, Wei-Jun Zhengb, Yong-Wei Liuc, 3
Jun Chena, Zhao-Shi Xua,3 and You-Zhi Maa,3 4
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Running title: Functional analysis of TaBZR2 in wheat 6
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1 This research was financially supported by the National Key Research and 8
Development Program of China (2016YFD0100600), the National Transgenic Key 9
Project of the Ministry of Agriculture of China (2018ZX0800909B), the National 10
Natural Science Foundation of China (31871624), and the Technological Innovation 11
Projects of Modern Agriculture of Hebei Province. 12
2 These authors contributed equally to the article. 13
3 Address correspondence to [email protected] or [email protected]. 14
15
The authors responsible for distribution of materials integral to the findings 16
presented in this article in accordance with the policy described in the instructions for 17
authors (www.plantphysiol.org) are: Zhao-Shi Xu ([email protected]) and You-Zhi 18
Ma ([email protected]). 19
Z.S.X. coordinated the project, conceived and designed experiments, and edited the 20
manuscript; X.Y.C. performed experiments and wrote the first draft of the manuscript; 21
Y.G. conducted the bioinformatic work and performed experiments; J.G., T.F.Y., 22
W.J.Z., and Y.W.L. generated and analyzed data; J.C. provided analytical tools and 23
managed reagents; Y.Z.M. coordinated the project. 24
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a Institute of Crop Science, Chinese Academy of Agricultural Sciences 26
(CAAS)/National Key Facility for Crop Gene Resources and Genetic Improvement, 27
Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of 28
Agriculture, Beijing 100081, China 29
Plant Physiology Preview. Published on March 6, 2019, as DOI:10.1104/pp.19.00100
Copyright 2019 by the American Society of Plant Biologists
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b College of Plant Protection/College of Agronomy, Northwest A&F University, 30
Yangling, Shaanxi 712100, China 31
c Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry 32
Sciences/Plant Genetic Engineering Center of Hebei Province, Shijiazhuang, Hebei 33
050051, China 34
d Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 35
266101, China. 36
37
Corresponding author: 38
39
40
Zhao-Shi Xu 41
Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS)/National 42
Key Facility for Crop Gene Resources and Genetic Improvement, Key Laboratory of 43
Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 44
100081, China 45
Telephone: +86-10-82106773 46
E-mail: [email protected] 47
48
You-Zhi Ma 49
Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS)/National 50
Key Facility for Crop Gene Resources and Genetic Improvement, Key Laboratory of 51
Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 52
100081, China 53
Telephone: +86-10-82109718 54
E-mail: [email protected] 55
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One-sentence summary: A BES/BZR-type transcription factor, TaBZR2, activates 57
TaGST1 to scavenge reactive oxygen species and mediates crosstalk between 58
brassinosteroids and drought signaling pathways. 59
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ABSTRACT 62
BRI1-EMS suppressor (BES)/brassinazole-resistant (BZR) family transcription 63
factors are involved in a variety of physiological processes, but the biological 64
functions of some BES/BZR transcription factors remain unknown; moreover, it is not 65
clear if any of these proteins function in the regulation of plant stress responses. Here, 66
TaBZR2-overexpressing plants exhibited drought tolerant phenotypes, whereas 67
down-regulation of TaBZR2 in wheat (Triticum aestivum) by RNA interference 68
resulted in elevated drought sensitivity. Electrophoretic mobility shift assay and 69
luciferase reporter analysis illustrate that TaBZR2 directly interacts with the gene 70
promoter to activate the expression of TaGST1, which functions positively in 71
scavenging drought-induced superoxide anions (O2-). Moreover, TaBZR2 acts as a 72
positive regulator in brassinosteroid (BR) signaling. Exogenous BR treatment 73
enhanced TaBZR2-mediated O2- scavenging and anti-oxidant enzyme gene expression. 74
Taken together, we demonstrate that a BES/BZR family transcription factor, TaBZR2, 75
functions positively in drought responses by activating TaGST1 and mediates the 76
crosstalk between BR and drought signaling pathways. Our results thus provide new 77
insights into the mechanisms underlying how BES/BZR family transcription factors 78
contribute to drought tolerance in wheat. 79
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INTRODUCTION 81
As sessile organisms, plants encounter various environmental stresses, such as 82
drought and salt stresses, that severely affect growth and productivity (Jeong et al., 83
2010; Takasaki et al., 2010; Yu et al., 2013; Zhang et al., 2017; Qi et al., 2018). Plants 84
have developed elaborate mechanisms to cope with such challenges via changes at the 85
physiological and biochemical levels as well as at the cellular and molecular levels 86
(Yamaguchi-Shinozaki and Shinozaki, 2006; Zhang et al., 2012; Yu et al., 2013; Liu et 87
al., 2018; Qi et al., 2018). These adaptive strategies are highly sophisticated processes 88
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regulated by an intricate signaling network and by orchestrating expression of 89
stress-responsive genes (Ramegowda et al., 2015; Liu et al., 2018; Wu et al., 2018). 90
Stress-responsive genes can be classified into two groups: effector genes and 91
regulatory genes (Huang et al., 2013; Liu et al., 2014; Kidokoro et al., 2015). 92
Effector genes encode enzymes required for osmoprotectants, late embryogenesis 93
abundant (LEA) proteins, aquaporin proteins, chaperones, and detoxification enzymes, 94
which protect cell membrane integrity, control ion balances, and scavenge reactive 95
oxygen species (ROS) (Huang et al., 2013; Liu et al., 2014). Regulatory genes encode 96
protein kinases and transcription factors, which function in signal perception, signal 97
transduction, and transcriptional regulation of gene expression (Huang et al., 2013; 98
Liu et al., 2014). Transcription factors, such as the dehydration responsive 99
element-binding (DREB)/C-repeat binding factor (CBF) family (Liu et al., 2013; 100
Kidokoro et al., 2015; Liu et al., 2018), APETALA2 (AP2)/ethylene responsive factor 101
(ERF) family (Seo et al., 2010; Rong et al., 2014), myeloblastosis (MYB) family (Li 102
et al., 2009; Seo et al., 2009; Seo et al., 2011), NAM, ATAF, and CUC (NAC) family 103
(Hao et al., 2011; Mao et al., 2015; Wang et al., 2018), WRKY family (Zhou et al., 104
2008; Wang et al., 2015), and basic leucine zipper (bZIP) family (Tang et al., 2012; 105
Song et al., 2013; Ma et al., 2018), can bind to cis-regulatory elements to modulate 106
the expression of various downstream genes, ultimately regulating adaptive responses 107
to unfavorable environmental conditions. 108
BRI1-EMS suppressor (BES)/brassinazole-resistant (BZR) transcription factors 109
form a small plant-specific gene family (Wang et al., 2002; Yin et al., 2005; Bai et al., 110
2007). Members of the BES/BZR family of transcription factors, which function 111
redundantly in BR response, are key components of the BR signaling pathway (Wang 112
et al., 2002; Yin et al., 2002; Yin et al., 2005; Li et al., 2010). BES1 and BZR1 are two 113
well-known BES/BZR family members that function as positive regulators in 114
Arabidopsis (Arabidopsis thaliana) BR signaling. Gain-of-function mutants bes1-D 115
and bzr1-1D can partially suppress the dwarf phenotypes of brassinosteroid 116
insensitive1 (bri1) and are resistant to the BR biosynthesis inhibitor brassinazole 117
(BRZ) (Wang et al., 2002; Yin et al., 2002). OsBZR1 functions as a positive regulator 118
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in the rice (Oryza sativa) BR signaling pathway, and 14-3-3 proteins inhibit OsBZR1 119
nuclear accumulation to negatively regulate BR signaling (Bai et al., 2007). 120
GmBEHL1 mediates the crosstalk between BR signaling and nodulation signaling 121
pathways that negatively regulates symbiotic nodulation in soybean (Glycine max) 122
(Yan et al., 2018). 123
In addition to their essential roles in BR signaling, BES/BZR family members 124
have been shown to function in Arabidopsis responses to drought and to stress from 125
both high and low temperatures (Oh et al., 2012; Li et al., 2017; Ye et al., 2017). The 126
drought-induced transcription factor RD26 mediates crosstalk between BR and 127
drought pathways via reciprocal inhibition between RD26 and BES1 transcriptional 128
activities (Ye et al., 2017). BZR1-PIF4 interaction integrates BR signaling and 129
environmental signals (Oh et al., 2012). BZR1 positively regulates Arabidopsis 130
freezing tolerance via DREB/CBF-dependent and DREB/CBF-independent pathways 131
(Li et al., 2017). 132
Bread wheat (Triticum aestivum L.) is a cereal crop that is widely grown throughout 133
the world. Drought profoundly affects wheat growth and productivity worldwide. 134
Although a few BES/BZR family members have been characterized in model plants, 135
the biological functions of wheat BES/BZR family transcription factors remain 136
largely unknown. In the present study, both drought and exogenous BR treatments 137
induced expression of a BES/BZR family transcription factor gene, TaBZR2. We then 138
analyzed the function of TaBZR2 through generating overexpression and RNA 139
interference (RNAi) transgenic wheat plants. Moreover, electrophoretic mobility shift 140
assay (EMSA) and luciferase reporter analysis illustrated that TaBZR2 functions 141
positively in drought tolerance by directly up-regulating the transcriptional activity of 142
wheat glutathione S-transferase 1 (TaGST1). Furthermore, TaBZR2 acts as a link 143
between BR and drought signaling pathways. 144
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RESULTS 147
Identification of Stress-Responsive BES/BZR Transcription Factors in Wheat 148
In previous whole-transcriptome analyses of drought and BR on wheat, the transcript 149
of TraesCS3D02G139300.1 was induced by both drought and exogenous BR 150
treatments and exhibited the greatest stress-inducible gene response (Supplemental 151
Table S1). Sequence alignment analysis revealed that this transcript encodes a protein 152
that shows high sequence similarity with rice BES/BZR transcription factor OsBZR2 153
(~ 87%) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We thus named this transcript 154
TaBZR2 and selected it for further analysis of its role in drought responses. 155
Protein structure analysis illustrated that the TaBZR2 amino acid sequence 156
contained an N-terminal DNA binding domain and 29 putative GSK3-like kinase 157
phosphorylation sites (S/TXXXS/T) but no putative PEST domain (a region rich in 158
proline, glutamate, serine, and threonine) 159
(http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind) or potential 14-3-3 binding 160
site (RXXXpSXP, where X is any amino acid, R is arginine, pS is phosphoserine, and P 161
is proline) was identified (Rechsteiner and Rogers, 1996; Wang et al., 2002; Yin et al., 162
2002; Bai et al., 2007) (Supplemental Fig. S1A). To explore the relationships among 163
wheat BZRs and their orthologs from other plant species, a phylogenetic tree was 164
constructed by amino acid sequence alignment. TaBZR2 was classified into subgroup 165
V, and the BES/BZRs derived from monocots clustered separately from those of 166
dicots, suggesting a potential functional diversity between dicot and monocot plants 167
(Supplemental Fig. S1B). 168
169
Drought and Exogenous BR Induced TaBZR2 Expression and the Nuclear 170
Accumulation of TaBZR2 Protein 171
We confirmed the expression patterns of TaBZR2 in drought and BR responses by 172
reverse transcription quantitative PCR (RT-qPCR) and immunoblot assays. Drought 173
induced TaBZR2 expression in both shoots and roots , reaching a peak at 2 h (Fig. 1, A 174
and B). TaBZR2 expression increased after treatment with exogenous BR and peaked 175
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at 4 h in BR-treated leaves and roots (Fig. 1, A and B). Furthermore, drought and 176
exogenous BR treatments increased the abundance of TaBZR2 protein (Fig. 1, C and 177
D). To better understand the biological functions of TaBZR2, we investigated the 178
subcellular localization of TaBZR2 protein in response to drought and exogenous BR 179
treatments. The TaBZR2-GFP fluorescence signal was observed in both the cytoplasm 180
and nucleus under unstressed conditions (Fig. 1E). Upon drought and exogenous BR 181
treatments, TaBZR2 proteins translocated from the cytoplasm to the nucleus as shown 182
by the nuclear/cytoplasmic signal ratio (Fig. 1E). 183
184
Overexpression of TaBZR2 Significantly Improves Drought Tolerance in 185
Transgenic Wheat 186
To investigate the drought tolerance associated with TaBZR2, we generated 187
transgenic bread wheat plants on the Fielder background in which TaBZR2, driven by 188
the maize (Zea mays) Ubiquitin promoter, was overexpressed. Three independent 189
transgenic lines that exhibited high TaBZR2 expression level based on RT-qPCR 190
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assays were chosen for further analysis (Fig. 2B). No differences were observed 191
between the TaBZR2-overexpressing (OE5, OE9, and OE11) and wild-type (WT) 192
plants under normal growth conditions (Fig. 2A). Drought treatment caused obvious 193
differences in growth and physiology of both TaBZR2-overexpressing and WT plants. 194
Upon drought treatment, compared with control plants, TaBZR2-overexpressing plants 195
had significantly delayed leaf rolling and higher survival rates (Fig. 2, A and C). 196
Moreover, the proline contents were significantly higher in TaBZR2-overexpressing 197
plants than in WT plants under drought conditions (Fig. 2D). The 198
TaBZR2-overexpressing plants had significantly lower electrolyte leakage levels and 199
malondialdehyde (MDA) contents compared to WT plants under drought conditions 200
(Fig. 2, E and F). Thus, TaBZR2 regulated physiological processes that improve the 201
drought tolerance of transgenic wheat plants. 202
203
Suppression of TaBZR2 Enhances Drought Sensitivity in Wheat 204
To further explore the function of TaBZR2 in drought responses, we produced two 205
independent TaBZR2-RNAi lines (Ri3 and Ri7) and determined the expression of 206
TaBZR2 using RT-qPCR assays. The expression levelof TaBZR2 decreased in the two 207
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lines (Fig. 3B and Supplemental Fig. S2), implying that TaBZR2 was successfully 208
suppressed. There were no obvious differences in the growth performance and 209
physiology between TaBZR2-RNAi and WT plants under normal growth conditions 210
(Fig. 3A). However, upon drought treatment, the survival rates were significantly 211
lower in TaBZR2-RNAi plants than in WT plants under drought conditions (Fig. 3C). 212
Moreover, drought-treated TaBZR2-RNAi lines had significantly lower proline 213
contents, higher electrolyte leakage levels, and higher MDA contents compared to WT 214
plants under drought conditions (Fig. 3, D–F). 215
216
TaBZR2 Positively Regulates the Expression of Multiple Stress-Related Genes 217
To explore how TaBZR2 contributes to drought tolerance, we performed RNA-Seq 218
assays to evaluate the differential gene expression between TaBZR2-overexpressing 219
and WT wheat plants under both normal and drought conditions. As shown in Figure 220
4A, using a threshold of a 2-fold change and a Student's t-test significance cut-off of P 221
<0.05, a comparison of the RNA-Seq data from TaBZR2-overexpressing and WT 222
plants under normal conditions identified 1,399 up-regulated and 1,064 223
down-regulated genes in TaBZR2-overexpressing plants (TaBZR2-OEN) compared 224
with those in WT plants (WTN). Upon drought treatment, the expression of 728 and 225
1,496 genes in the TaBZR2-overexpressing plants (TaBZR2-OED) was up- or 226
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down-regulated, respectively, compared with that in WT plants (WTD). In total, 227
20,224 differentially expressed genes (DEGs) were identified between drought-treated 228
and normal growth WT plants (WTD/WTN). Cluster and Venn diagram analyses 229
revealed that the expression patterns of all DEGs in TaBZR2-OED compared with that 230
in WTD (TaBZR2-OED/WTD) did not significantly overlap with TaBZR2-OEN 231
compared with WTN (TaBZR2-OEN/WTN), or WTD compared with WTN 232
(WTD/WTN). These results demonstrated that TaBZR2 significantly affects the 233
global gene expression profile in wheat, indicating that unknown mechanisms may 234
underlie the drought tolerance of transgenic wheat. 235
Gene Ontology (GO) analysis revealed that the DEGs between the drought-treated 236
TaBZR2-overexpressing and WT plants were significantly enriched in biological 237
process categories including "response to abiotic stimulus", "response to water stress", 238
and "regulation of metabolic and biosynthetic processes" (Fig. 4B). Interestingly, we 239
found that the expression of a range of well-known stress-related genes were among 240
the up-regulated DEGs for the drought-treated TaBZR2-overexpressing plants 241
(Supplemental Table S2). Note that we also used RT-qPCR assays to successfully 242
verify the up-regulated expression trends for the genes identified from the RNA-Seq 243
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data, including TaGST1, TaLEA3, TaDHN3, TaP5CS, TaPOD21, and TaSAPK3 (Fig. 244
4C). Consistent with a direct functional impact of TaBZR2 on the expression of these 245
known stress-related genes, we also used RT-qPCR to examine the expression of 246
these genes in the aforementioned drought-treated TaBZR2-RNAi plants and found 247
that their expression was significantly reduced compared to both drought-treated 248
TaBZR2-RNAi and WT plants (Fig. 4C). GST genes, encoding ROS-scavenging 249
enzymes, function in protecting plants against oxidative damage under stress 250
conditions (Jha et al., 2011; Rong et al., 2014). P5CS is the key enzyme for proline 251
synthesis (Yoshiba et al., 1995; Zhuo et al., 2017). Dehydrins are responsive to 252
various environmental stresses and exhibit multiple biochemical activities, such as 253
buffering water, sequestering ions, stabilizing membranes, or acting as chaperones 254
(Kovacs et al., 2008; Tang et al., 2012; Rong et al., 2014; Zhuo et al., 2017). Sucrose 255
non-fermenting-1-related protein kinase 2 (SnRK2) is implicated in stress signaling 256
transduction via abscisic acid (ABA)-dependent and -independent pathways (Yoshida 257
et al., 2002; Zhang et al., 2011), TaBZR2 could modulate the expression of numerous 258
stress-responsive genes under drought conditions, contributing to the drought 259
tolerance of the transgenic wheat. 260
261
TaBZR2 Functions Positively in Scavenging Drought-Induced Superoxide Anions 262
(O2-) 263
Environmental stimuli, including drought, salt, and high and low temperatures, 264
induce the accumulation of toxic ROS, including H2O2 and O2-, which if not 265
controlled, can eventually lead to oxidative damage (Dat et al., 2000; Wang et al., 266
2017). TaBZR2 has a role in activating antioxidant enzyme gene expression. To 267
investigate whether TaBZR2 participates in scavenging ROS, we analyzed the ROS 268
contents between TaBZR2-RNAi and WT wheat lines under normal and drought 269
conditions. There was no significant difference in H2O2 accumulation between 270
TaBZR2-RNAi and WT wheat lines under unstressed and drought conditions 271
(Supplemental Fig. S3). The O2- contents of TaBZR2-RNAi and WT wheat lines were 272
similar under unstressed conditions (Fig. 5, A and B). Nevertheless, the O2- contents 273
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were significantly higher in the TaBZR2-RNAi plants than in the WT plants under 274
drought conditions (Fig. 5, A and B). DMTU acts as a O2- scavenging reagent (Lv et 275
al., 2018). When DMTU was added to the growth medium to reduce O2-, the O2- 276
contents of TaBZR2-RNAi wheat lines recovered to a level similar to that of the WT 277
plants (Fig. 5, A and B). 278
To investigate whether the TaBZR2-mediated O2- scavenging was associated with 279
the positive role of TaBZR2 in drought responses, we compared the biomass of 280
TaBZR2-RNAi wheat plants with that of the WT wheat plants grown on 1/2-strength 281
Hoagland’s nutrient solution supplemented with different concentrations of PEG 6000 282
and DMTU (0, 15% PEG 6000, 1 mM DMTU, and 15% PEG 6000 + 1 mM DMTU). 283
Biomass was similar for the WT and TaBZR2-RNAi plants grown on 1/2-strength 284
Hoagland’s nutrient solution containing 0 and 1 mM DMTU (Fig. 5, C and D). 285
However, biomass was significantly larger in the WT plants than in the 286
TaBZR2-RNAi plants under drought conditions (Fig. 5, C and D). DMTU treatment 287
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can partially suppress drought (15% PEG 6000)-induced biomass reduction. 288
Importantly, the biomass of TaBZR2-RNAi plants was comparable with that of the 289
WT plants grown on 1/2-strength Hoagland’s nutrient solution containing 15% PEG 290
6000 and 1 mM DMTU (Fig. 5, C and D). These results indicate that TaBZR2 has a 291
role in scavenging O2- to alleviate drought stress. 292
293
TaBZR2 Functions as a Positive Regulator of TaGST1 Expression by Binding to 294
Its Promoter and Activating Transcription 295
RNA-Seq and RT-qPCR analyses both indicated that the expression of TaGST1 is 296
up-regulated by TaBZR2 overexpression, so the transcription of this gene may be 297
activated by TaBZR2. Previous studies have revealed that BES/BZR family 298
transcription factors can bind to E-box (5’-CANNTG-3’) cis-elements to regulate the 299
expression of target genes; we detected ten E-box cis-elements in the TaGST1 300
promoter. We thus used EMSAs to investigate whether TaBZR2 can directly bind to 301
the TaGST1 promoter in vitro. The EMSAs showed that the TaBZR2-GST fusion 302
protein was able to bind to the TaGST1 promoter; no such binding was observed for 303
the control GST protein (Fig. 6, A and B). Further, the observed binding to the 304
biotin-labeled target sequences was dramatically reduced when unlabeled competitor 305
target DNA sequences were added, and no binding was detected when adding the 306
mutated biotin-labeled TaGST1 probes (Fig. 6, A and B). Having determined that 307
TaBZR2 can bind the TaGST1 promoter in vitro, we next used a wheat protoplast 308
transient expression system to assess whether this binding can drive TaGST1 gene 309
expression in vivo. A pGreen II 0800 vector harboring a LUC reporter gene driven by 310
the TaGST1 promoter (~2000-bp) was co-transformed into wheat protoplasts 311
transfected with an empty pJIT16318 vector or a pJIT16318-TaBZR2 vector. 312
Compared with the empty-vector control samples, the protoplasts expressing TaBZR2 313
exhibited significantly increased expression of the reporter (Fig. 6C). To further test if 314
the activation effect of TaBZR2 on the TaGST1 was through binding to the E-box 315
(CACGTG, -1475 to -1481), the TaGST1 promoter containing the mutated E-box was 316
inserted into the pGreen II 0800 vector and coexpressed with the pJIT16318-TaBZR2 317
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vector in wheat protoplasts. The results demonstrated that the E-box mutation 318
(AAAAAA, -1475 to -1481) disrupted TaBZR2-mediated activation of the TaGST1 319
promoter (Fig. 6C), indicating that the TaBZR2 transcription factor is a positive 320
regulator of TaGST1 expression. 321
322
Overexpression of TaGST1 Significantly Improved Drought Tolerance in 323
Transgenic Wheat by Reducing O2- Contents 324
To investigate the function of TaGST1 in the drought response, we generated 325
transgenic wheat plants that overexpressed TaGST1 under the control of the maize 326
Ubiquitin promoter. Three independent homozygous T3 transgenic lines with 327
relatively high expression of TaGST1 were selected for additional phenotypic analyses 328
(Fig. 7E). Under normal growth conditions, there were no notable differences in plant 329
growth or physiology between TaGST1-overexpressing (OE1, OE4, and OE9) and 330
WT plants. However, upon drought treatment, the survival rate of 331
TaGST1-overexpressing plants was significantly higher than that of WT plants (Fig. 7, 332
A and B). Moreover, the drought-treated TaGST1-overexpressing plants had 333
significantly lower O2- content compared to WT plants (Fig. 7, C and D). 334
335
TaBZR2 is a Positive Regulator in the BR Signaling Pathway 336
To obtain more detailed evidence for the role of TaBZR2 in BR responses, we 337
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transformed the BR-insensitive mutant bri1-5 with a TaBZR2 overexpression 338
construct under the control of the CaMV 35S promoter. Two independent homozygous 339
T3 transgenic lines (35S:TaBZR2/bri1-5-B3 and -B7) that strongly expressed TaBZR2 340
were selected for further phenotypic analysis. Overexpression of TaBZR2 partially 341
suppressed the dwarf phenotypes of bri1-5 mutant plants (Supplemental Fig. S4, A 342
and B). Compared with bri1-5 mutant plants, 35S:TaBZR2/bri1-5 transgenic plants 343
had enhanced tolerance to the BR biosynthetic inhibitor BRZ (Supplemental Fig. 344
S4B). In addition, compared with bri1-5 mutant plants, 35S:TaBZR2/bri1-5 transgenic 345
plants showed reduced expression of the BR biosynthesis genes CPD and DWF4 and 346
increased expression of the BR signaling gene SAUR-AC (Supplemental Fig. S4C). 347
To obtain further insights into the role of TaBZR2 in the wheat BR signaling 348
pathway, we investigated the BR sensitivity of TaBZR2 transgenic wheat plants. 349
TaBZR2-overexpressing and TaBZR2-RNAi wheat plants exhibited altered BR 350
sensitivity as indicated by their root length in the absence or presence of BR. In the 351
absence of BR, there was no significant difference in root lengths between 352
TaBZR2-overexpressing, TaBZR2-RNAi, and WT wheat plants (Fig. 8A). However, in 353
the presence of BR, the root lengths of TaBZR2-overexpressing lines were shorter than 354
that of WT plants. Moreover, compared with WT plants, TaBZR2-RNAi plants 355
exhibited BR-insensitive phenotypes with longer roots (Fig. 8A). Previous studies 356
have shown that BES/BZR family transcription factors can bind to the BR-response 357
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element (BRRE) in the promoters of feedback-regulated BR biosynthetic genes to 358
repress their expression (He et al., 2005). EMSAs demonstrated that TaBZR2 can bind 359
to the BRRE cis-regulatory elements in the promoter of the BR biosynthentic gene 360
TaD2 (Fig. 8B). Furthermore, RT-qPCR assays revealed that, compared with WT 361
plants, TaBZR2-overexpressing plants showed reduced expression of the BR 362
biosynthetic genes TaD2 and TaDWARF, whereas TaBZR2-RNAi plants showed 363
enhanced expression of the BR biosynthetic genes TaD2 and TaDWARF in the 364
absence and presence of BR (Fig. 8C). The TaBZR2-modulated inhibition of TaD2 365
and TaDWARF expressions were larger in the presence of BR than in the absence of 366
BR (Fig. 8C). Our data suggest that TaBZR2 functions as a positive regulator in BR 367
signaling. 368
369
TaBZR2 is Involved in BR-mediated Drought Responses 370
To investigate whether TaBZR2 has a role in BR-mediated drought responses, we 371
investigated the expression of stress responsive genes encoding antioxidant enzymes, 372
including TaGST1, TaPOD21, and TaDHN3, in response to BR treatment by RT-qPCR 373
analyses. Upon exogenous BR treatment, the expression of these genes in 374
TaBZR2-overexpression plants was enhanced compared to WT plants, whereas their 375
expression in TaBZR2-RNAi plants was reduced under normal and drought conditions 376
(Fig. 9A). In addition, drought and BR treatments enhanced the abundance of 377
dephosphorylated TaBZR2 proteins in the TaBZR2-overexpressing, TaBZR2-RNAi, 378
and WT plants (Fig. 9B). The amounts of dephosphorylated TaBZR2 proteins were 379
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17
larger in TaBZR2-overexpression plants than in WT plants. Nevertheless, compared to 380
WT plants, the amount of dephosphorylated TaBZR2 proteins in TaBZR2-RNAi plants 381
was smaller upon drought and BR treatments (Fig. 9B). The phosphorylation status of 382
the BES/BZR family transcription factors is usually used as the biochemical maker 383
for BR signaling outputs (Zhang et al., 2009). Treatment of immunoprecipitated 384
protein with protein phosphatase eliminated the slowly migrating band (Supplemental 385
Fig. S5), strongly suggesting that the fast band is unphosphorylated and the slow band 386
is phosphorylated TaBZR2. In addition, the O2- accumulation of 387
TaBZR2-overexpressing, TaBZR2-RNAi, and WT plants was similar under normal 388
conditions. When exposed to induced drought conditions, O2- accumulation increased 389
in the roots of TaBZR2-overexpressing, TaBZR2-RNAi, and WT plants. The O2- 390
contents of TaBZR2-overexpressing plants under drought conditions was significantly 391
lower than that of WT plants, whereas the O2- accumulation was significantly higher in 392
TaBZR2-RNAi plants than in WT plants (Fig. 9, C and D). Exogenous BR treatment 393
repressed the O2- accumulation in wheat plants under normal and drought conditions. 394
Compared to WT plants, the BR-mediated O2- scavenging was enhanced in 395
TaBZR2-overexpressing plants, whereas BR-mediated O2- scavenging was reduced in 396
TaBZR2-RNAi plants under drought conditions (Fig. 9, C and D). These results 397
indicated that TaBZR2 participates in BR-mediated O2- scavenging. 398
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399
400
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19
DISCUSSION 401
Plant genomes have many kinds of transcription factors that function importantly in 402
plant adaption to extreme environmental conditions (Zhang et al., 2017; Liu et al., 403
2018; Ma et al., 2018). BES/BZR proteins constitute another important family of 404
plant-specific transcription factors (Wang et al., 2002; Yin et al., 2002; Yin et al., 405
2005), but comparatively little is known about their biological functions in drought 406
responses. In the present study, a drought-inducible BES/BZR-type transcription 407
factor gene TaBZR2 was identified from wheat drought transcriptome data, and 408
follow-up work illustrated that overexpression of TaBZR2 enhanced drought tolerance 409
in transgenic wheat plants with larger accumulation of osmoprotectant metabolites, 410
higher membrane stability, and lower ROS contents compared with the control plants 411
under drought conditions, whereas TaBZR2-RNAi wheat lines exhibited the opposite 412
trend. These results suggest that TaBZR2 functions positively in regulating drought 413
responses in wheat. 414
BES/BZR family members regulate the expression of target genes by interacting 415
with BRRE and/or or E-box cis-elements in their promoters (Goda et al., 2004; 416
Nemhauser et al., 2004; He et al., 2005; Wang et al., 2006; Walcher and Nemhauser, 417
2012; Li et al., 2017). For example, BZR1 binds to BRRE and/or E-box elements in 418
the promoters of the genes encoding CBF1/DREB1A, CBF2/DREB1B, and WRKY6 419
to modulate their expression, contributing to freezing tolerance in Arabidopsis (Li et 420
al., 2017), and BES1 directly binds to the E-box element of the SAUR-AC15 promoter 421
to enhance auxin signaling in Arabidopsis (Goda et al., 2004; Walcher and Nemhauser, 422
2012). Our EMSA and luciferase reporter analyses demonstrated that TaBZR2 directly 423
binds to the promoter of TaGST1 to activate its transcription. GST genes encode 424
detoxification enzymes that function in maintaining cell redox homeostasis and 425
protecting organisms against oxidative stress under stress conditions (Jha et al., 2011; 426
Rong et al., 2014; Qi et al., 2018). Our data illustrated that, compared with the WT 427
plants, the TaGST1-overexpressing wheat lines exhibited drought tolerance 428
phenotypes with lower O2- contents under drought conditions, which was consistent 429
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20
with the positive role of TaBZR2 in scavenging drought-induced O2-. These results 430
indicate that TaBZR2 has a role in activating TaGST1 for scavenging O2-, and 431
consequently alleviate drought stress. 432
Genetic and molecular studies have greatly increased our understanding of the BR 433
signaling pathway in model plants (Bai et al., 2007; Yu et al., 2008; Ye et al., 2011; 434
Chen et al., 2017). BRs are perceived by a leucine-rich repeat (LRR)-receptor kinase, 435
BRI1, and transduces the signal to activate the BES/BZR family transcription factor, 436
which regulates the expression of a large number of genes (Wang et al., 2002; Yin et 437
al., 2002; Yin et al., 2005; Bai et al., 2007; Oh et al., 2012; Jiang et al., 2013; Shimada 438
et al., 2015; Yan et al., 2018). Consistent with the positive role of BES/BZR family 439
members in the BR signaling pathway (Wang et al., 2002; Yin et al., 2002; Yan et al., 440
2018), TaBZR2 positively regulates BR signaling in wheat. Exogenous application of 441
BR protects plants from drought stress (Kagale et al., 2007; Xia et al., 2009; Divi et 442
al., 2010, 2016; Nawaz et al., 2017). Previous studies have shown that some 443
components of the BR signaling pathway are involved in drought responses (Koh et 444
al., 2007; Sahni et al., 2016). Overexpression of the Arabidopsis BR biosynthetic gene 445
DWARF4 confers drought tolerance in Brassica napus (Sahni et al., 2016). OsGSK1 is 446
a negative regulator of rice BR signaling: its T-DNA knockout mutants display 447
enhanced tolerance to drought and other abiotic stresses (Koh et al., 2007). 448
Considering that TaBZR2 functions as a positive regulator in drought responses, it is 449
possible that TaBZR2 has a role in mediating the crosstalk between BR and drought 450
responses. A recent study has shown that BR is involved in regulation of the 451
accumulation of O2- (Lv et al., 2018). For example, the BR-deficient mutant det2-9 452
accumulates more O2- in roots (Lv et al., 2018). Our data demonstrated that the 453
expression of some TaBZR2 target genes encoding antioxidant enzymes, including 454
TaGST1, TaPOD21, and TaDHN3, was up-regulated upon exogenous BR treatment. 455
Furthermore, exogenous application of BR can enhance TaBZR2-mediated activation 456
of antioxidant enzymes and scavenging of O2- under drought conditions. Our data 457
indicated that TaBZR2 participates in BR-mediated drought response partially by 458
reducing the accumulation of O2-. 459
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21
It is worth noting that recent studies also illustrated several components of the BR 460
signaling pathway negatively regulate drought responses (Chen et al., 2017; Ye et al., 461
2017). For example, in contrast to a positive role of TaBZR2 in drought responses, 462
AtBES1 negatively regulates plant drought tolerance (Ye et al., 2017). BES/BZR 463
family transcription factor genes derived from monocots clustered separately from 464
those of dicots by phylogenetic analysis. Protein structure analyses illustrated that the 465
amino acid sequence of TaBZR2 has an N-terminal binding domain and GSK3-like 466
kinase phosphorylation sites, but no 14-3-3 binding domain and PEST motif were 467
identified, which was different from the well-known BES/BZR family members like 468
AtBES1, AtBZR1, and OsBZR1 (Wang et al., 2002; Yin et al., 2002; Bai et al., 2007). 469
Furthermore, TaBZR2 exhibited a different BR regulated mobility shift pattern with 470
AtBZR1 and AtBES1. Previously, studies revealed that all of phosphorylated AtBZR1 471
and AtBES1 were dephosphorylated upon BR treatment (He et al., 2002; Yin et al., 472
2002), whereas BR treatment caused partially phosphorylated TaBZR2 to convert to 473
the dephosphorylated form. These results indicate that although BES/BZR family 474
members function positively in BR signaling, protein structural differences and the 475
different mechanisms of action may lead to functional differences in environmental 476
stress responses. Our study expands the known functional scope of the BES/BZR 477
family members, and its basic insights should inform the work of both plant abiotic 478
stress researchers and wheat breeders and biotechnologists. 479
480
MATERIALS AND METHODS 481
Plant Materials and Growth Conditions 482
Wheat (Triticum aestivum) plants used for molecular analysis were grown in a 483
greenhouse at 70% relative humidity, 25/23°C day/night temperatures, and long-day 484
conditions (16 h light/8 h dark photoperiod) with a light intensity of approximately 485
300 μmol m-2 s-1. The wheat cultivar KeNong 199 was used to amplify cDNA 486
sequences of TaBZR2 and TaGST1. The wheat cultivar Fielder was used as the 487
receptor material to generate transgenic plants. To analyze the expression of TaBZR2 488
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22
under abiotic stress conditions, wheat cultivar KeNong 199 seedlings were grown in 489
1/2-strength Hoagland’s liquid medium in a greenhouse with 70% relative humidity, 490
25/23°C day/night temperatures, long-day conditions (16 h light/8 h dark photoperiod), 491
with a light intensity of approximately 400 μmol m-2 s-1 for 2 weeks. For BR and 492
drought stress treatments, the roots of wheat seedlings were immersed in 1/2-strength 493
Hoagland’s solution containing 1 μM EBL solution (Sigma-Aldrich, USA) and 15% 494
(w/v) PEG 6000. Leaves and roots were sampled at 0, 1, 2, 4, 8, 12, and 24 h, and then 495
immediately frozen in liquid nitrogen and stored at -80°C prior to RNA extraction. The 496
Arabidopsis (Arabidopsis thaliana) plants were subsequently grown in a greenhouse 497
at 23°C under long-day conditions (16 h light/8 h dark photoperiod) and a light 498
intensity of approximately 100 μmol m-2 s-1. For Arabidopsis, the seeds were 499
germinated on 1/2-strength Murashige and Skoog (MS) (Caisson Labs, USA) media 500
supplemented with 2% (w/v) sucrose and grown for a week, after which the seedlings 501
were transplanted into soil. The plants were subsequently grown in a greenhouse with 502
70% relative humidity, 23°C, and long-day conditions (16 h light/8 h dark 503
photoperiod) with a light intensity of approximately 100 μmol m-2 s-1 for 3 weeks. The 504
Arabidopsis BR-insensitive mutant bri1-5 was used for transformation. 505
506
Generation of Transgenic Arabidopsis and Wheat 507
To generate TaBZR2 transgenic wheat plants, the coding region( CDS) of TaBZR2D 508
were cloned into the plant transformation vector pWMB110 driven by the maize (Zea 509
mays) Ubiquitin promoter. The 198-bp TaBZR2 specific fragment was synthesized by 510
Beijing AuGCT Company, which was then fused in both sense and antisense 511
orientations to flank the 508-bp rice (Oryza sativa) zinc finger type family protein gene 512
intron 6. This recombinant DNA was then inserted into the pWMB110 vector to 513
generate the pWMB110-TaBZR2-RNAi construct. To generate TaGST1-overexpression 514
wheat plants, the TaGST1 CDS were also inserted into the pMWB110 vector, driven by 515
the maize Ubiquitin promoter. Genetic transformations were performed using an 516
Agrobacterium tumefaciens-mediated transformation system. To isolate positive 517
transgenic wheat lines, leaves of 10-day-old transgenic wheat seedlings grown in 518
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23
1/2-strength Hoagland’s nutrient solution were used for RNA isolation, and then RT- 519
and RT-qPCR analyzes were performed. For Arabidopsis, the CDS of TaBZR2 was 520
introduced into the plant transformation vector pBI121 under the control of the CaMV 521
35S promoter. The resultant constructs were confirmed by sequencing and then 522
transformed into BR-insensitive mutant bri1-5 plants via the vacuum infiltration 523
method (Bechtold and Pelletier, 1998). Homozygous T3 seeds of the transgenic lines 524
were used for phenotypic analyses. Primers used in these studies are in Supplemental 525
Table S3. 526
527
Drought Stress Treatment 528
For drought tolerance assays, TaBZR2 transgenic and WT wheat seedlings were 529
planted in pots containing mixed soil (1:1 vermiculite:humus) and cultured normally in 530
the greenhouse for 3 weeks (until seedlings were at the 3-leaf stage), after which these 531
seedlings were deprived of water until significant differences in wilting were observed 532
between transgenic and WT wheat plants. Three independent experiments were 533
performed. TaGST1 transgenic and WT wheat seedlings were planted in pots 534
containing mixed soil (1:1 vermiculite:humus) and cultured normally in the greenhouse 535
for 3 weeks (until seedlings were at the 3-leaf stage), after which 15% (w/v) PEG 6000 536
solution was applied to the bottom of the plates for ~14 days until significant 537
differences in wilting were observed between transgenic and WT wheat plants. Three 538
independent experiments were performed. 539
540
BR Sensitivity Assays 541
For BR sensitivity assays, sterilized seeds of TaBZR2-overexpressing, 542
TaBZR2-RNAi, and WT wheat plants were maintained at 4°C for 1 week, after which 543
the germinated seeds were transplanted into 1/2-strength Hoagland’s solution 544
containing different concentrations of EBL (0, 0.25, and 1 μM). After 7 days of growth 545
at 23°C under long-day conditions (16-h light/8-h dark photoperiod), images were 546
taken, and the primary root length for each seedling was evaluated using an Epson 547
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24
Expression 11000XL root system scanning analyzer (Epson, Japan). For hypocotyl 548
elongation assays, the sterilized seeds of WT plants, 35S:TaBZR2/bri1-5 transgenic 549
Arabidopsis plants, and bri1-5 plants were sown on 1/2-strength MS growth media 550
supplemented with various concentrations (0, 0.25, and 0.5 μM) of BRZ and then kept 551
at 4°C in the dark for 3 days. After 7 days of growth at 22°C under dark conditions, 552
images were taken, and the lengths of hypocotyls were measured. 553
554
RNA Extraction and RT-qPCR Assays 555
The total RNA from Arabidopsis and wheat seedlings was extracted using Trizol 556
reagent (TaKaRa, Japan), and their DNA was digested using RNase-free DNaseI 557
(TaKaRa, Japan). First-strand cDNA was synthesized using a PrimeScript First-Strand 558
cDNA Synthesis Kit (TaKaRa, Japan). RT-qPCR was performed with an ABI 7500 559
real-time PCR system (ThermoFisher Scientific, USA) in conjunction with SYBR to 560
monitor double-stranded DNA products. The reaction was conducted at 95°C for 5 561
min, then 42 cycles of 95°C for 15 s, 58°C to 60°C for 25 s, and 72°C for 30 s. A 562
quantitative analysis using the 2-ΔΔCT method was subsequently performed (Le et al., 563
2011). Each experiment was performed with at least three independent biological 564
replicates. For each primer pair, the amplification efficiency was checked using a 565
melting-curve analysis. For wheat, β-actin was used as the internal control and actin 566
was used an internal control for Arabidopsis (Liu et al., 2013). The specific primers 567
used for RT-qPCR are listed in Supplemental Table S3. 568
569
Immunoblot Assay 570
Wheat cultivar KeNong 199 seedlings were grown in 1/2-strength Hoagland’s liquid 571
medium in a greenhouse with 70% relative humidity, 25/23°C day/night temperatures, 572
and long-day conditions (16 h light/8 h dark photoperiod) with a light intensity of 573
approximately 400 μmol m-2 s-1 for 2 weeks. For BR and drought stress treatments, the 574
roots of wheat seedlings were immersed in 1/2-strength Hoagland’s solution containing 575
1 μM EBL solution and 15% (w/v) PEG 6000. Leaves were sampled at 0, 2, 4, 8, and 576
12 h and then used to extract total protein. Plant protein was isolated with lysis buffer 577
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25
(50 m M Tris [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10 m M MgCl2, 10% [v/v] 578
glycerol, 1 mM PMSF, 5 mM DTT, protease inhibitor cocktail Complete Minitablets 579
[Roche], and 0.2% [v/v] Nonidet P-40). For phosphatase treatment, the extracted plant 580
proteins were treated with the Lambda protein phosphatase (NEB, P0753S, USA) 581
according to the manufacturer’s instructions. The dephosphorylation reaction took 582
place at 30°C for 30 min in a thermal cycler (Bio-Rad, USA). TaBZR2 proteins were 583
subsequently detected by immunoblotting using Anti-TaBZR2 antibodies at a 1:1000 584
dilution. IRDye 800CW anti-rabbit IG (H + L) at a 1:10000 dilution (LI-COR, USA) 585
was used as a second antibody. The immunoblots were developed via an Odyssey 586
CLx Infrared Imaging System (LI-COR, USA). 587
588
Subcellular Localization 589
Transient expression assays were conducted as described previously (Liu et al., 590
2013). TaBZR2 was inserted into the subcellular localization vector pJIT16318, which 591
contains a CaMV 35S promoter and a C-terminal GFP. Approximately 4 × 104 592
mesophyll protoplasts were isolated from 10-day-old wheat seedlings and then 593
transfected with pJIT16318-TaBZR2 plasmids by PEG-mediated transformation. The 594
transfected protoplasts were then incubated at 23°C for 12 h. GFP fluorescence in the 595
transformed protoplasts was imaged using a confocal laser-scanning microscope (LSM 596
700, Germany). 597
598
Measurements of Proline Content, Electrolyte Leakage Level, and MDA Content 599
For assays of physiological traits, 3-week-old wheat seedlings at the 3 leaf stage 600
were treated with drought conditions for ~ 12 d. About 0.2 g of wheat leaf leaves were 601
harvested for measurements of physiological parameters. Absorbance values were 602
measured with a Varioskan LUX Multimode Microplate Reader (ThermoFisher 603
Scientific, USA). The proline concentration was determined as described previously 604
(Zhang et al., 2012). The electrolyte leakage was examined in accordance with 605
previously described methods (Cao et al., 2007), and the MDA content was assayed as 606
described previously (Zhang et al., 2012). All of the measurements were repeated three 607
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26
times. 608
609
Measurements of O2- Content and H2O2 Content 610
To investigate the contents of O2- and H2O2, 2-week-old wheat seedlings grown on 611
1/2-strength Hoagland’s nutrient solution supplemented with different concentrations 612
of PEG 6000 and BR (0, 15% (w/v) PEG 6000, 10 nM BR, 15% (w/v) PEG 6000 + 613
10 nM BR, 1 mM DMTU, and 15% (w/v) PEG 6000 + 1 mM DMTU) for 72 h. The 614
O2- contents were measured following the protocol of the Superoxide anion content 615
detection kit (Solarbio, USA, BC1295). The H2O2 contents were measured following 616
the protocol of the H2O2 content detection kit (Solarbio, BC3595, USA). For 617
Nitro-blue tetrazolium (NBT) staining, the wheat roots were immersed in NBT stain 618
solution for 30 min and the dark blue color appeared following the protocol of the 619
Alkaline Phosphatase Activity Detection Kit (Amersco, USA). The staining reaction 620
was stopped by the addition of an excess of 95% ethanol. Images were observed and 621
photographed under a stereomicroscope (Leica, Germany). 622
623
RNA-Seq Assays 624
TaBZR2-overexpressing (OE9) and WT (Fielder) plants were grown in 1/2-strength 625
Hoagland’s liquid medium in a greenhouse with 70% relative humidity, 25/23°C 626
day/night temperatures, and long-day conditions (16 h light/8 h dark photoperiod) with 627
a light intensity of approximately 400 μmol m-2 s-1 for 2 weeks. Then, the wheat 628
seedlings were transferred to fresh 1/2-strength Hoagland’s solution that contained 15% 629
(w/v) PEG 6000. Leaves were sampled at 0 and 6 h for transcriptome sequencing 630
experiments, and three biological replicates were used. The RNA-Seq analysis was 631
performed by the Allwegene Company (Beijing). Total RNA was extracted from the 632
samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s 633
instructions, and RNA sequencing was conducted on an Illumina HiSeq platform. 634
RNA sequencing data were analyzed as previously described (Mortazavi et al., 2008). 635
DEGs were selected using DESeq (1.10.1) with a relative change threshold of 2-fold 636
(P < 0.05, false discovery rate < 0.01) (Anders and Huber, 2010). GO categories were 637
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27
identifined using the GOseq R package (Young et al., 2010). The genome annotation 638
and functional categorization are based on the National Center for Biotechnology 639
Information non-redundant protein sequences 640
(https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/). 641
642
EMSA Assay 643
The CDS of TaBZR2 was inserted into the pGEX-4T-1 vector. The GST and GST- 644
TaBZR2 fusion proteins were expressed in Escherichia coli (BL21) and purified by 645
glutathione-Sepharose TM 4B (GE Healthcare, Sweden) according to the 646
manufacturer’s protocol. The biotin-labeled probes used in this assay were 647
synthesized (AuGCT, china), and the sequences are listed in Supplemental Table S3. 648
Double-stranded DNA was obtained by heating oligonucleotides at 95 °C for 10 min 649
and annealing at room temperature. The EMSA assay was performed using the 650
LightShift Chemiluminescent EMSA Kit (Thermo, USA) according to the 651
manufacturer’s instructions. In brief, 2 mg of purified fusion protein GST-TaBZR2 or 652
GST protein was added to the binding reaction. The binding reaction took place at 653
25°C for 30 min in a thermal cycler (Bio-Rad, USA). The mixture was separated on a 654
6% polyacrylamide mini gel, and then the DNA was transferred to nylon membrane 655
(Millipore, USA). The signal was visualized with an EasySee Western Blot Kit 656
(TransGen, china). 657
658
Transcriptional Activation Assays in Wheat Protoplasts 659
For the transcriptional activation assay, the promoter fragment of TaGST1 was 660
inserted into LUC reporter plasmid pGreen II 0800, which contained a Renilla 661
luciferase (REN) gene under the control of the CaMV 35S promoter used as an 662
internal control. The effector plasmids and the reporter plasmids were co-transformed 663
into protoplasts by PEG-mediated transformation. After culturing for 16 h at 23°C, the 664
activities of LUC and REN were separately determined using a Dual-Luciferase 665
Reporter Assay System (Promega, E1910, USA). 666
667
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28
Antibody Preparation 668
Anti-TaBZR2 was generated by Wuhan Abclonal Biotechnology Cooperation. 669
TaBZR2 CDs (453–999 bp) were inserted into the pET32a vector. Purified 670
His-TaBZR2 (151–333 amino acids) fusion protein was injected into rabbits to 671
produce TaBZR2 polyclonal antibodies. Immunoblots were performed using 672
antiserum against TaBZR2 and visualized with an EasySee Western Blot Kit 673
(TransGen). 674
675
Accession Numbers 676
RNA sequencing data described in this study can be found in the National Center 677
for Biotechnology Information Sequence Read Archive 678
(http://www.ncbi.nlm.nih.gov/sra) under accession number SRP071191. 679
680
Supplemental Data 681
The following supplemental materials are available. 682
Supplemental Figure S1. Sequence and phylogenetic analyses of TaBZR2. 683
684
Supplemental Figure S2. The expression level of BES/BZR family transcription 685
factor genes in TaBZR2-RNAi and WT wheat plants. 686
687
Supplemental Figure S3. Measurements of H2O2 contents in TaBZR2-overexpressing, 688
TaBZR2-RNAi, and WT wheat plants under normal and drought conditions. 689
690
Supplemental Figure S4. TaBZR2 overexpression partially rescued the dwarf 691
phenotypes of bri1-5 plants. 692
693
Supplemental Figure S5. Immunoblot analysis of TaBZR2 protein. 694
695
Supplemental Table S1. Wheat BZRs responsive to drought and BR treatments. 696
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29
697
Supplemental Table S2. Analysis of stress-related genes in 698
TaBZR2-overexpressing and WT wheat plants under drought conditions. 699
700
Supplemental Table S3. Primers and probes used in this study. 701
702
ACKNOWLEDGEMENTS 703
We are grateful to Drs. Rui-Lian Jing and Yong-Fu Fu (Institute of Crop Science, 704
Chinese Academy of Agricultural Sciences) for providing wheat seeds and for the 705
BiFC system, respectively. We also thank Dr. Dongying Gao (Department of Plant 706
Sciences, University of Georgia, USA) for suggestions on the manuscript. 707
708
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30
FIGURE LEGENDS 709
Figure 1 Expression and localization of TaBZR2 in wheat under BR and drought 710
conditions. (A) and (B) The expression profile of TaBZR2 in 2-week-old wheat 711
seedling leaf and root tissue under drought and BR treatments for the indicated time. 712
Transcript levels were quantified by RT-qPCR assays. The expression of β-actin was 713
analyzed as internal control. Each data point is the mean (± SE) of three experiments. 714
(C) and (D) Protein level of TaBZR2 in 2-week-old wheat seedlings after drought and 715
BR treatments for the indicated time. Total proteins were extracted and subjected to 716
immunoblot analysis with anti-TaBZR2 antibodies. Rubisco was used as a loading 717
control. (E) Localization of TaBZR2 protein under drought and BR conditions. The 718
nuclear/cytoplasmic signal ratio represents nuclear-accumulated TaBZR2 versus 719
cytoplasmic-accumulated TaBZR2. Images were observed under a laser scanning 720
confocal microscope. Scale bar = 12 μm. Each data point is the mean (± SE) of ten 721
biological replicates (**P < 0.01; Student’s t-test). 722
723
Figure 2 TaBZR2-overexpressing wheat plants exhibit improved drought tolerance. 724
(A) Phenotypes of TaBZR2-overexpressing (OE5, OE9, and OE11) and wild-type 725
(WT) wheat plants under well-watered and drought conditions. (B) RT-qPCR analysis 726
of TaBZR2 gene expression in TaBZR2-overexpressing and WT plants. The expression 727
of β-actin was analyzed as an internal control. Each data point is the mean (± SE) of 728
three experiments. (C) Survival rate of the control and water-stressed plants (without 729
irrigation for 21 d). (D) Proline content in seedlings under normal and drought 730
conditions. (E) Electrolyte leakage in seedlings under normal and drought conditions. 731
(F) MDA content in seedlings under normal and drought conditions. Each data point 732
is the mean (± SE) of three experiments (10 seedlings per experiment). The asterisks 733
indicate significant differences between TaBZR2-overexpressing and WT wheat plants 734
(Student’s t-test, *P < 0.05 and **P < 0.01). 735
736
Figure 3 TaBZR2-RNAi wheat plants show enhanced drought sensitivity. (A) 737
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31
Phenotypes of TaBZR2-RNAi (Ri3 and Ri7) and WT wheat plants under well-watered 738
and drought conditions. (B) RT-qPCR analysis of TaBZR2 gene expression in 739
TaBZR2-RNAi and WT plants. The expression of β-actin was analyzed as an internal 740
control. Each data point is the mean (± SE) of three experiments. (C) Survival rate of 741
the control and water-stressed plants (without irrigation for 18 d). (D) Proline content 742
in seedlings under normal and drought conditions. (E) Electrolyte leakage in seedlings 743
under normal and drought conditions. (F) MDA content in seedlings under normal and 744
drought conditions. Each data point is the mean (± SE) of three experiments (10 745
seedlings per experiment). The asterisks indicate significant differences between 746
TaBZR2-RNAi and WT wheat plants (Student’s t-test, *P < 0.05 and **P < 0.01). 747
748
Figure 4 Analysis of the expression levels of TaBZR2 downstream genes. (A) Venn 749
diagrams comparing the up- and down-regulated genes between WT plants under 750
normal and drought conditions (WTN and WTD), and TaBZR2-overexpressing and 751
WT plants under normal (TaBZR2-OEN/WTN) and drought conditions 752
(TaBZR2-OED/WTD). (B) Functional categorization analysis of candidate TaBZR2 753
target genes in biological process under drought conditions. (C) The expression levels 754
of drought-responsive genes in TaBZR2-overexpressing, TaBZR2-RNAi, and WT 755
wheat plants. Two-week-old wheat seedlings treated with 15% PEG 6000 for 6 h were 756
used for RNA isolation. Transcript levels were quantified by RT-qPCR assays, and the 757
expression of β-actin was used as an internal control. Each data point is the mean (± 758
SE) of three experiments (10 seedlings per experiment). 759
760
Figure 5 TaBZR2 functions positively in savenging drought-induced O2-. (A) NBT 761
staining in primary root tips of TaBZR2-RNAi and WT wheat plants grown in 762
1/2-strength Hoagland’s liquid medium, medium containing 15% PEG 6000, medium 763
containing 1 mM DMTU, or medium containing 15% PEG 6000 + 1 mM DMTU for 72 764
h. The strength of color shows the concentration of O2- in the root tips. Scale bar = 1 765
mm (B) Measurements of the O2- contents of TaBZR2-RNAi and WT wheat plants 766
grown in 1/2-strength Hoagland’s liquid medium, medium containing 15% PEG 6000, 767
www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
32
medium containing 1 mM DMTU, or medium containing 15% PEG 6000 + 1 mM 768
DMTU for 72 h. Each data point is the mean (± SE) of six biological replicates. The 769
asterisks indicate significant differences between TaBZR2-RNAi and WT wheat plants 770
(Student’s t-test, **P < 0.01) (C) Phentypes of TaBZR2-RNAi and WT wheat plants 771
grown in 1/2-strength Hoagland’s liquid medium, medium containing 15% PEG 6000, 772
medium containing 1 mM DMTU, or medium containing 15% PEG 6000 + 1 mM 773
DMTU. Scale bar = 2 cm. (D) Measurement of the total fresh weight of TaBZR2-RNAi 774
and WT wheat plants grown in 1/2-strength Hoagland’s liquid medium, medium 775
containing 15% PEG 6000, medium containing 1 mM DMTU, or medium containing 776
15% PEG 6000 + 1 mM DMTU. Each data point is the mean (± SE) of six biological 777
replicates. The asterisks indicate significant differences between TaBZR2-RNAi and 778
WT wheat plants (Student’s t-test, *P < 0.05). 779
780
Figure 6 TaBZR2 directly regulates the expression of TaGST1. (A) The diagram 781
shows the structure of the TaGST1 promoter. The sequences represent TaGST1 probe 782
sequences. The underlined sequences indicated the core elements or mutated core 783
elements in the TaGST1 probe. (B) EMSA assay of TaBZR2 binding to the promoter 784
of TaGST1. Biotin-labeled TaGST1 probes (normal and mutated) were incubated with 785
GST or GST-TaBZR2 protein. 100× competitor fragments were added to analyze the 786
specificity of binding. (C) TaBZR2 increases TaGST1 promoter activity in wheat 787
protoplasts. TaBZR2 was co-transfected with either TaGST1 promoter or mutated 788
TaGST1 promoter. The LUC to REN ratio is shown and indicates the activity of the 789
transcription factors on the expression level of the promoters. Each data point is the 790
mean (± SE) of ten biological replicates (**P < 0.01; Student’s t-test). 791
792
Figure 7 TaGST1 overexpression promotes drought tolerance in transgenic wheat. (A) 793
Phenotypes of TaGST1-overexpressing and WT plants under normal and drought 794
conditions. (B) Survival rate of control and water-stressed plants (15% PEG 6000 795
treatment for 14 d). Each data point is the mean (± SE) of three experiments (10 796
seedlings per experiment). (C) NBT staining in primary root tip of 797
www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
33
TaGST1-overexpressing and WT seedlings with 0 or 15% PEG 6000 treatment for 72 798
h. The strength of color shows the concentration of O2- in the root tips. Scale bar =1 799
mm (D) Measurements of the O2- contents of TaGST1-overexpressing and WT plants 800
under normal and drought conditions. Each data point is the mean (± SE) of six 801
biological replicates. (E) RT-qPCR analysis of TaGST1 gene expression in 802
TaGST1-overexpressing and WT wheat seedlings. The expression of β-actin was used 803
as an internal control. Each data point is the mean (± SE) of three experiments (10 804
seedlings per experiment). The asterisks indicate significant differences between 805
TaGST1-overexpressing and WT plants (Student’s t-test, **P < 0.01). 806
807
Figure 8 TaBZR2 is a positive regulator in the BR signaling pathway. (A) Phenotypes 808
of TaBZR2-overexpressing (OE5, OE9, and OE11), TaBZR2-RNAi (Ri3 and Ri7), and 809
WT wheat plants grown in 1/2-strength Hoagland’s liquid medium or medium 810
containing 1 μM BR. Bar = 2 cm. Root length of TaBZR2-overexpressing, 811
TaBZR2-RNAi, and WT wheat plants grown on 1/2-strength Hoagland’s medium that 812
contained different concentrations of BR (0, 0.25, or 1 μM) in the light for 7 d. Each 813
data point is the mean (± SE) of three experiments (20 seedlings per experiment). The 814
asterisks indicate significant differences between TaBZR2 transgenic 815
(TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and WT plants (Student’s 816
t-test, *P < 0.05). (B) EMSA assay of TaBZR2 binding to the BR-response elements 817
(BRRE) in the promoter of TaD2. Biotin-labeled BRRE probes (normal and mutated) 818
were incubated with GST or GST-TaBZR2 protein. 100× competitor fragments were 819
added to analyze the specificity of binding. (C) The expression levels of BR 820
biosynthetic genes in TaBZR2 transgenic (TaBZR2-overexpressing lines and 821
TaBZR2-RNAi lines) and WT plants. The expression of β-actin was used as an internal 822
control. Each data point is the mean (± SE) of three experiments (10 seedlings per 823
experiment). 824
825
Figure 9 TaBZR2 regulates wheat drought tolerance through the BR-dependent 826
pathway. (A) The expression levels of stress-responsive genes in TaBZR2 transgenic 827
www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
34
(TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and WT plants grown in 828
1/2-strength Hoagland’s liquid medium, medium containing 15% PEG 6000, medium 829
containing 10 nM BR, or medium containing 15% PEG 6000 + 10 nM BR for 6 h. Each 830
data point is the mean (± SE) of three experiments (10 seedlings per experiment). (B) 831
Protein level of TaBZR2 in TaBZR2-overexpressing, TaBZR2-RNAi, and WT wheat 832
plants upon drought and BR treatments for 6 h. Total proteins were extracted and 833
subjected to immunoblot analysis with anti-TaBZR2 antibodies. Rubisco was used as 834
a loading control. (C) NBT staining in primary root tip of TaBZR2-overexpressing, 835
TaBZR2-RNAi, and WT wheat plants grown in 1/2-strength Hoagland’s liquid 836
medium, medium containing 15% PEG 6000, medium containing 10 nM BR, or 837
medium containing 15% PEG 6000 + 10 nM BR for 72 h. The strength of color shows 838
the concentration of O2- in the root tips. Scale bar = 1 mm (D) Measurements of the 839
O2- contents of TaBZR2-overexpressing, TaBZR2-RNAi, and WT wheat plants grown 840
in 1/2-strength Hoagland’s liquid medium, medium containing 15% PEG 6000, 841
medium containing 10 nM BR, or medium containing 15% PEG 6000 + 10 nM BR for 842
72 h. Each data point is the mean (± SE) of six biological replicates. The asterisks 843
indicate significant differences between TaBZR2 transgenic (TaBZR2-overexpressing 844
lines and TaBZR2-RNAi lines) and WT plants (Student’s t-test, **P <0.01). 845
846
847
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Parsed CitationsAnders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11: R106
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bai MY, Zhang LY, Gampala SS, Zhu SW, Song WY, Chong K, Wang ZY (2007) Functions of OsBZR1 and 14-3-3 proteins inbrassinosteroid signaling in rice. Proc Natl Acad Sci USA 104: 13839–13844
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuuminfiltration. Methods Mol Biol 82: 259–266
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cao WH, Liu J, He XJ, Mu RL, Zhou HL, Chen SY, Zhang JS (2007) Modulation of ethylene responses affects plant salt-stressresponses. Plant Physiol 143: 707–719
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen J, Nolan TM, Ye H, Zhang M, Tong H, Xin P, Chu J, Chu C, Li Z, Yin Y (2017) Arabidopsis WRKY46, WRKY54, and WRKY70transcription factors are involved in brassinosteroid-regulated plant growth and drought responses. Plant Cell 29: 1425–1439
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van Breusegem F (2000) Dual action of the active oxygen species duringplant stress responses. Cell Mol Life Sci 57: 779–795
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Divi UK, Rahman T, Krishna P (2010) Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid,ethylene and salicylic acid pathways. BMC Plant Biol 10: 151
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Divi UK, Rahman T, Krishna P (2016) Gene expression and functional analyses in brassinosteroid-mediated stress tolerance. PlantBiotechnol J 14: 419–432
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Goda H, Sawa S, Asami T, Fujioka S, Shimada Y, Yoshida S (2004) Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis. Plant Physiol 134: 1555–1573
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hao YJ, Wei W, Song QX, Chen HW, Zhang YQ, Wang F, Zou HF, Lei G, Tian AG, Zhang WK, Ma B, Zhang JS, Chen SY (2011) SoybeanNAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant J 68: 302–313
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
He JX, Gendron JM, Sun Y, Gampala SSL, Gendron N, Sun CQ, Wang ZY (2005) BZR1 is a transcriptional repressor with dual roles inbrassinosteroid homeostasis and growth responses. Science 307: 1634–1638
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
He JX, Gendron JM, Yang YL, Li JM, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positiveregulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci USA 99: 10185–10190
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Huang XS, Wang W, Zhang Q, Liu JH (2013) A basic helix-loop-helix transcription factor, PtrbHLH, of Poncirus trifoliata confers coldtolerance and modulates peroxidase-mediated scavenging of hydrogen peroxide. Plant Physiol 162: 1178–1194
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jeong JS, Kim YS, Baek KH, Jung H, Ha SH, Do Choi Y, Kim M, Reuzeau C, Kim JK (2010) Root-specific expression of OsNAC10improves drought tolerance and grain yield in rice under field drought conditions. Plant Physiol 153: 185–197
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jha B, Sharma A, Mishra A (2011) Expression of SbGSTU (tau class glutathione S-transferase) gene isolated from Salicornia brachiatain tobacco for salt tolerance. Mol Biol Rep 38: 4823–4832
Pubmed: Author and Title www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Jiang JJ, Zhang C, Wang XL (2013) Ligand perception, activation, and early signaling of plant steroid receptor brassinosteroidinsensitive 1. J Integr Plant Biol 55: 1198–1211
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kagale S, Divi UK, Krochko JE, Keller WA, Krishna P (2007) Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassicanapus to a range of abiotic stresses. Planta 225: 353–364
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kidokoro S, Watanabe K, Ohori T, Moriwaki T, Maruyama K, Mizoi J, Htwe NMPS, Fujita Y, Sekita S, Shinozaki K, Yamaguchi-ShinozakiK (2015) Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive geneexpression. Plant J 81: 505–518
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Koh S, Lee SC, Kim MK, Koh JH, Lee S, An G, Choe S, Kim SR (2007) T-DNA tagged knockout mutation of rice OsGSK1, an orthologueof Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses. Plant Mol Biol 65: 453–466
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kovacs D, Kalmar E, Torok Z, Tompa P (2008) Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins.Plant Physiol 147: 381–390
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Le DT, Nishiyama R, Watanabe Y, Mochida K, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS (2011) Genome-wide expression profilingof soybean two-component system genes in soybean root and shoot tissues under dehydration stress. DNA Res 18: 17–29
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li H, Ye K, Shi Y, Cheng J, Zhang X, Yang S (2017) BZR1 positively regulates freezing tolerance via CBF-dependent and CBF-independent pathways in Arabidopsis. Mol Plant 10: 545–559
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li L, Ye H, Guo H, Yin Y (2010) Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormonebrassinosteroid regulated gene expression. Proc Natl Acad Sci USA 107: 3918–3923
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li L, Yu XF, Thompson A, Guo M, Yoshida S, Asami T, Chory J, Yin Y (2009) Arabidopsis MYB30 is a direct target of BES1 andcooperates with BES1 to regulate brassinosteroid-induced gene expression. Plant J 58: 275–286
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu JY, Shi YT, Yang SH (2018) Insights into the regulation of C-repeat binding factors in plant cold signaling. J Integr Plant Biol 60:780–795
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu P, Xu ZS, Lu PP, Hu D, Chen M, Li LC, Ma YZ (2013) A wheat PI4K gene whose product possesses threonine autophophorylationactivity confers tolerance to drought and salt in Arabidopsis. J Exp Bot 64: 2915–2927
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu SX, Wang XL, Wang HW, Xin HB, Yang XH, Yan JB, Li JS, Tran LSP, Shinozaki K, Yamaguchi-Shinozaki K, Qin F (2013) Genome-wide analysis of ZmDREB genes and their association with natural variation in drought tolerance at seedling stage of Zea mays L. PloSGenet 9: e1003790
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu WW, Tai HH, Li SS, Gao W, Zhao M, Xie CX, Li WX (2014) bHLH122 is important for drought and osmotic stress resistance inArabidopsis and in the repression of ABA catabolism. New Phytol 201: 1192–1204
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lv B, Tian H, Zhang F, Liu J, Lu S, Bai M, Li C, Ding Z (2018) Brassinosteroids regulate root growth by controlling reactive oxygenspecies homeostasis and dual effect on ethylene synthesis in Arabidopsis. PLoS Genet 14: e1007144
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ma H, Liu C, Li Z, Ran Q, Xie G, Wang B, Fang S, Chu J, Zhang J (2018) ZmbZIP4 contributes to stress resistance in maize by regulating www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
ABA synthesis and root development. Plant Physiol 178: 753–770Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mao HD, Wang HW, Liu SX, Li Z, Yang XH, Yan JB, Li JS, Tran LSP, Qin F (2015) A transposable element in a NAC gene is associatedwith drought tolerance in maize seedlings. Nat Commun 6: 8326
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. NatMethods 5: 621–628
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nawaz F, Naeem M, Zulfiqar B, Akram A, Ashraf MY, Raheel M, Shabbir RN, Hussain RA, Anwar I, Aurangzaib M (2017) Understandingbrassinosteroid-regulated mechanisms to improve stress tolerance in plants: a critical review. Environ Sci Pollut Res Int 24: 15959–15975
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nemhauser JL, Mockler TC, Chory J (2004) Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol 2: E258Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Oh E, Zhu JY, Wang ZY (2012) Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat CellBiol 14: 802–809
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Qi J, Song CP, Wang B, Zhou J, Kangasjarvi J, Zhu JK, Gong Z (2018) Reactive oxygen species signaling and stomatal movement inplant responses to drought stress and pathogen attack. J Integr Plant Biol 60: 805–826
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ramegowda V, Basu S, Gupta C, Pereira A (2015) Regulation of grain yield in rice under well-watered and drought stress conditions byGUDK. Plant Signal Behav 10: e1034421
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rechsteiner M, Rogers SW (1996) PEST sequences and regulation by proteolysis. Trends Biochem Sci 21: 267–271Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rong W, Qi L, Wang A, Ye X, Du L, Liang H, Xin Z, Zhang Z (2014) The ERF transcription factor TaERF3 promotes tolerance to salt anddrought stresses in wheat. Plant Biotechnol J 12: 468–479
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sahni S, Prasad BD, Liu Q, Grbic V, Sharpe A, Singh SP, Krishna P (2016) Overexpression of the brassinosteroid biosynthetic geneDWF4 in Brassica napus simultaneously increases seed yield and stress tolerance. Sci Rep-UK 6: 28298
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Seo PJ, Lee SB, Suh MC, Park MJ, Go YS, Park CM (2011) The MYB96 transcription factor regulates cuticular wax biosynthesis underdrought conditions in Arabidopsis. Plant Cell 23: 1138–1152
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Seo PJ, Xiang FN, Qiao M, Park JY, Lee YN, Kim SG, Lee YH, Park WJ, Park CM (2009) The MYB96 transcription factor mediatesabscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol 151: 275–289
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Seo YJ, Park JB, Cho YJ, Jung C, Seo HS, Park SK, Nahm BH, Song JT (2010) Overexpression of the ethylene-responsive factor geneBrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants. Mol Cells 30: 271–277
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shimada S, Komatsu T, Yamagami A, Nakazawa M, Matsui M, Kawaide H, Natsume M, Osada H, Asami T, Nakano T (2015) Formation anddissociation of the BSS1 protein complex regulates plant development via brassinosteroid signaling. Plant Cell 27: 375–390
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Song QX, Li QT, Liu YF, Zhang FX, Ma B, Zhang WK, Man WQ, Du WG, Wang GD, Chen SY, Zhang JS (2013) Soybean GmbZIP123 geneenhances lipid content in the seeds of transgenic Arabidopsis plants. J Exp Bot 64: 4329–4341 www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from
Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Takasaki H, Maruyama K, Kidokoro S, Ito Y, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K, Nakashima K (2010) The abiotic stress-responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress tolerance in rice. Mol Genet Genomics284: 173–183
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tang N, Zhang H, Li X, Xiao J, Xiong L (2012) Constitutive activation of transcription factor OsbZIP46 improves drought tolerance inrice. Plant Physiol 158: 1755–1768
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Walcher CL, Nemhauser JL (2012) Bipartite promoter element required for auxin response. Plant Physiol 158: 273–282Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang B, Wei J, Song N, Wang N, Zhao J, Kang Z (2018) A novel wheat NAC transcription factor, TaNAC30, negatively regulatesresistance of wheat to stripe rust. J Integr Plant Biol 60: 432–443
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang F, Chen HW, Li QT, Wei W, Li W, Zhang WK, Ma B, Bi YD, Lai YC, Liu XL, Man WQ, Zhang JS, Chen SY (2015) GmWRKY27interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants. Plant J 83: 224–236
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang N, Zhang W, Qin M, Li S, Qiao M, Liu Z, Xiang F (2017) Drought tolerance conferred in soybean (Glycine max. L) by GmMYB84, anovel R2R3-MYB transcription factor. Plant Cell Physiol 58: 1764–1776
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang ZY, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, Chory J (2002) Nuclear-localizedBZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2: 505–513
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang ZY, Wang QM, Chong K, Wang FR, Wang L, Bai MY, Jia CG (2006) The brassinosteroid signal transduction pathway. Cell Res 16:427–434
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wu Q, Wang M, Shen J, Chen D, Zheng Y, Zhang W (2018) ZmOST1 mediates abscisic acid regulation of guard cell ion channels anddrought stress responses. J Integr Plant Biol
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xia XJ, Wang YJ, Zhou YH, Tao Y, Mao WH, Shi K, Asami T, Chen ZX, Yu JQ (2009) Reactive oxygen species are involved inbrassinosteroid-induced stress tolerance in cucumber. Plant Physiol 150: 801–814
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydrationand cold stresses. Annu Rev Plant Biol 57: 781–803
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yan QQ, Wang LX, Li X (2018) GmBEHL1, a BES1/BZR1 family protein, negatively regulates soybean nodulation. Sci Rep-UK 8: 7614Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ye HX, Li L, Yin YH (2011) Recent advances in the regulation of brassinosteroid signaling and biosynthesis pathways. J Integr PlantBiol 53: 455–468
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ye HX, Liu SZ, Tang BY, Chen JN, Xie ZL, Nolan TM, Jiang H, Guo HQ, Lin HY, Li L, Wang YQ, Tong HN, Zhang MC, Chu CC, Li ZH,Aluru M, Aluru S, Schnable PS, Yin YH (2017) RD26 mediates crosstalk between drought and brassinosteroid signalling pathways. NatCommun 8: 14573
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J (2005) A new class of transcription factors mediates brassinosteroid-regulatedgene expression in Arabidopsis. Cell 120: 249–259 www.plantphysiol.orgon February 19, 2020 - Published by Downloaded from
Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yin Y, Wang ZY, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J (2002) BES1 accumulates in the nucleus in response tobrassinosteroids to regulate gene expression and promote stem elongation. Cell 109: 181–191
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, Yamaguchishinozaki K, Wada K, Harada Y, Shinozaki K (1995) Correlationbetween the induction of a gene for delta(1)-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thalianaunder osmotic-stress. Plant J 7: 751–760
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshida R, Hobo T, Ichimura K, Mizoguchi T, Takahashi F, Aronso J, Ecker JR, Shinozaki K (2002) ABA-activated SnRK2 protein kinaseis required for dehydration stress signaling in Arabidopsis. Plant Cell Physiol 43: 1473–1483
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol11: R14
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yu L, Chen X, Wang Z, Wang S, Wang Y, Zhu Q, Li S, Xiang C (2013) Arabidopsis enhanced drought tolerance1/HOMEODOMAINGLABROUS11 confers drought tolerance in transgenic rice without yield penalty. Plant Physiol 162: 1378–1391
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yu X, Li L, Li L, Guo M, Chory J, Yin Y (2008) Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containingproteins ELF6 and REF6 in Arabidopsis. Proc Natl Acad Sci USA 105: 7618–7623
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang H, Mao X, Jing R, Chang X, Xie H (2011) Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved inabiotic stress responses. J Exp Bot 62: 975–988
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang L, Zhao G, Xia C, Jia J, Liu X, Kong X (2012) A wheat R2R3-MYB gene, TaMYB30-B, improves drought stress tolerance intransgenic Arabidopsis. J Exp Bot 63: 5873–5885
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang N, Yin Y, Liu X, Tong S, Xing J, Zhang Y, Pudake RN, Izquierdo EM, Peng H, Xin M, Hu Z, Ni Z, Sun Q, Yao Y (2017) The E3 ligaseTaSAP5 alters drought stress responses by promoting the degradation of DRIP proteins. Plant Physiol 175: 1878–1892
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang SS, Cai ZY, Wang XL (2009) The primary signaling outputs of brassinosteroids are regulated by abscisic acid signaling. Proc NatlAcad Sci USA 106: 4543–4548
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang ZY, Liu X, Wang XD, Zhou MP, Zhou XY, Ye XG, Wei XN (2012) An R2R3 MYB transcription factor in wheat, TaPIMP1, mediateshost resistance to Bipolaris sorokiniana and drought stresses through regulation of defense- and stress-related genes. New Phytol196: 1155-1170
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhou QY, Tian AG, Zou HF, Xie ZM, Lei G, Huang J, Wang CM, Wang HW, Zhang JS, Chen SY (2008) Soybean WRKY-type transcriptionfactor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsisplants. Plant Biotechnol J 6: 486–503
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhuo C, Liang L, Zhao Y, Guo Z, Lu S (2017) A cold responsive ethylene responsive factor from Medicago falcata confers coldtolerance by up-regulation of polyamine turnover, antioxidant protection, and proline accumulation. Plant Cell Environ 41: 2021–2032.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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