BES/BZR Transcription Factor TaBZR2 Positively Regulates … · BRI1-EMS suppressor (BES)/brassinazole-resistant (BZR) family transcription factors are involved in a variety of physiological

BES/BZR Transcription Factor TaBZR2 PositivelyRegulates Drought Responses by Activationof TaGST11[OPEN]

Xiao-Yu Cui,a,d,2 Yuan Gao,a,2 Jun Guo,b Tai-Fei Yu,a Wei-Jun Zheng,b Yong-Wei Liu,c Jun Chen,a

Zhao-Shi Xu,a,3,4 and You-Zhi Maa,3

aInstitute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility for Crop GeneResources and Genetic Improvement, Key Laboratory of Biology and Genetic Improvement of Triticeae Crops,Ministry of Agriculture, Beijing 100081, ChinabCollege of Plant Protection/College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100,ChinacInstitute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences/Plant GeneticEngineering Center of Hebei Province, Shijiazhuang, Hebei 050051, ChinadTobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China

ORCID ID: 0000-0001-8028-6413 (X.-Y.C.).

BRI1-EMS suppressor (BES)/brassinazole-resistant (BZR) family transcription factors are involved in a variety of physiologicalprocesses, but the biological functions of some BES/BZR transcription factors remain unknown; moreover, it is not clear if any ofthese proteins function in the regulation of plant stress responses. Here, wheat (Triticum aestivum) brassinazole-resistant 2(TaBZR2)-overexpressing plants exhibited drought tolerant phenotypes, whereas downregulation of TaBZR2 in wheat byRNA interference resulted in elevated drought sensitivity. electrophoretic mobility shift assay and luciferase reporter analysisillustrate that TaBZR2 directly interacts with the gene promoter to activate the expression of T. aestivum glutathiones-transferase-1 (TaGST1), which functions positively in scavenging drought-induced superoxide anions (O2

2). Moreover,TaBZR2 acts as a positive regulator in brassinosteroid (BR) signaling. Exogenous BR treatment enhanced TaBZR2-mediatedO2

2 scavenging and antioxidant enzyme gene expression. Taken together, we demonstrate that a BES/BZR family transcriptionfactor, TaBZR2, functions positively in drought responses by activating TaGST1 and mediates the crosstalk between BR anddrought signaling pathways. Our results thus provide new insights into the mechanisms underlying how BES/BZR familytranscription factors contribute to drought tolerance in wheat.

As sessile organisms, plants encounter variousenvironmental stresses, such as drought and saltstresses, that severely affect growth and productivity

(Jeong et al., 2010; Takasaki et al., 2010; Yu et al., 2013;Zhang et al., 2017; Qi et al., 2018). Plants have devel-oped elaborate mechanisms to cope with such chal-lenges via changes at the physiological and biochemicallevels as well as at the cellular and molecular levels(Yamaguchi-Shinozaki and Shinozaki, 2006; Zhanget al., 2012b; Yu et al., 2013; Liu et al., 2018; Qi et al.,2018). These adaptive strategies are highly sophisti-cated processes regulated by an intricate signalingnetwork and by orchestrating expression of stress-responsive genes (Ramegowda et al., 2015; Liu et al.,2018; Wu et al., 2018). Stress-responsive genes can beclassified into two groups: effector genes and regula-tory genes (Huang et al., 2013; Liu et al., 2014; Kidokoroet al., 2015).Effector genes encode enzymes required for osmo-

protectants, late embryogenesis abundant proteins,aquaporin proteins, chaperones, and detoxificationenzymes, which protect cell membrane integrity, controlion balances, and scavenge reactive oxygen species(ROS; Huang et al., 2013; Liu et al., 2014). Regulatorygenes encode protein kinases and transcription factors,which function in signal perception, signal transduction,

1This work was supported by the National Key Research and De-velopment Program of China (grant no. 2016YFD0100600), the Na-tional Transgenic Key Project of the Ministry of Agriculture of China(grant no. 2018ZX0800909B), the National Natural Science Founda-tion of China (grant no. 31871624), and the Technological InnovationProjects of Modern Agriculture of Hebei Province.

2These authors contributed equally to this article.3Senior authors.4Author for contact: [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the instructions for authors (www.plantphysiol.org) is:Zhao-Shi Xu ([email protected]).

Z.S.X. coordinated the project, conceived and designed experi-ments, and edited the manuscript; X.Y.C. performed experimentsand wrote the first draft of the manuscript; Y.G. conducted the bio-informatic work and performed experiments; J.G., T.F.Y., W.J.Z., andY.W.L. generated and analyzed data; J.C. provided analytical toolsand managed reagents; Y.Z.M. coordinated the project.

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and transcriptional regulation of gene expression(Huang et al., 2013; Liu et al., 2014). Transcription fac-tors, such as the dehydration responsive element-binding (DREB)/C-repeat binding factor (CBF) family(Liu et al., 2013b, 2018; Kidokoro et al., 2015), APE-TALA2/ethylene responsive factor family (Seo et al.,2010; Rong et al., 2014), myeloblastosis family (Liet al., 2009; Seo et al., 2009, 2011), NAC (NAM,ATAF, and CUC) family (Hao et al., 2011; Mao et al.,2015; Wang et al., 2018), WRKY family (Zhou et al.,2008; Wang et al., 2015), and basic Leu zipper family(Tang et al., 2012; Song et al., 2013; Ma et al., 2018), canbind to cis-regulatory elements to modulate the ex-pression of various downstream genes, ultimately regu-lating adaptive responses to unfavorable environmentalconditions.

BRI1-EMS suppressor (BES)/brassinazole-resistant(BZR) transcription factors form a small plant-specificgene family (Wang et al., 2002; Yin et al., 2005; Bai et al.,2007). Members of the BES/BZR family of transcriptionfactors, which function redundantly in BR response,are key components of the BR signaling pathway(Wang et al., 2002; Yin et al., 2002, 2005; Li et al., 2010).BES1 and BZR1 are two well-known BES/BZRfamily members that function as positive regulators inArabidopsis (Arabidopsis thaliana) BR signaling. Gain-of-function mutants bes1-D and bzr1-1D can partiallysuppress the dwarf phenotypes of brassinosteroid in-sensitive1 (bri1) and are resistant to the BR biosynthesisinhibitor brassinazole (BRZ;Wang et al., 2002; Yin et al.,2002). OsBZR1 functions as a positive regulator in therice (Oryza sativa) BR signaling pathway, and 14-3-3proteins inhibit OsBZR1 nuclear accumulation to neg-atively regulate BR signaling (Bai et al., 2007).GmBEHL1 mediates the crosstalk between BR signal-ing and nodulation signaling pathways that negativelyregulates symbiotic nodulation in soybean (Glycinemax; Yan et al., 2018).

In addition to their essential roles in BR signaling,BES/BZR family members have been shown to func-tion in Arabidopsis responses to drought and to stressfrom both high and low temperatures (Oh et al., 2012; Liet al., 2017; Ye et al., 2017). The drought-induced tran-scription factor RD26 mediates crosstalk between BRand drought pathways via reciprocal inhibition betweenRD26 and BES1 transcriptional activities (Ye et al., 2017).BZR1-PIF4 interaction integrates BR signaling and en-vironmental signals (Oh et al., 2012). BZR1 positivelyregulates Arabidopsis freezing tolerance via DREB/CBF-dependent andDREB/CBF-independent pathways(Li et al., 2017).

Bread wheat (Triticum aestivum) is a cereal crop thatis widely grown throughout the world. Drought pro-foundly affects wheat growth and productivityworldwide. Although a few BES/BZR family mem-bers have been characterized in model plants, thebiological functions of wheat BES/BZR family tran-scription factors remain largely unknown. In thisstudy, both drought and exogenous BR treatmentsinduced expression of a BES/BZR family transcription

factor gene, TaBZR2. We then analyzed the function ofTaBZR2 through generating overexpression and RNAinterference (RNAi) transgenic wheat plants. More-over, electrophoretic mobility shift assay (EMSA) andluciferase (LUC) reporter analysis illustrated thatTaBZR2 functions positively in drought tolerance bydirectly upregulating the transcriptional activity of T.aestivum glutathione s-transferase-1 (TaGST1). Fur-thermore, TaBZR2 acts as a link between BR anddrought signaling pathways.

RESULTS

Identification of Stress-Responsive BES/BZR TranscriptionFactors in Wheat

In previous whole-transcriptome analyses of droughtand BR onwheat, the transcript of TraesCS3D02G139300.1was induced by both drought and exogenous BR treat-ments and exhibited the greatest stress-inducible generesponse (Supplemental Table S1). Sequence alignmentanalysis revealed that this transcript encodes a proteinthat shows high sequence similarity with rice BES/BZRtranscription factor OsBZR2 (;87%; https://blast.ncbi.nlm.nih.gov/Blast.cgi). We thus named this transcriptTaBZR2 and selected it for further analysis of its role indrought responses.

Protein structure analysis illustrated that theTaBZR2 amino acid sequence contained an N-terminalDNA binding domain and 29 putative glycogen syn-thase kinase3 (GSK3)-like kinase phosphorylation sites(S/TXXXS/T) but no putative PEST domain (a re-gion rich in Pro, Glu, Ser, and Thr; http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind) or po-tential 14-3-3 binding site (RXXXpSXP, where X is anyamino acid, R is Arg, pS is phospho-Ser, and P is Pro)was identified (Rechsteiner and Rogers, 1996;Wang et al., 2002; Yin et al., 2002; Bai et al., 2007;Supplemental Fig. S1A). To explore the relationshipsamong wheat BZRs and their orthologs from otherplant species, a phylogenetic tree was constructed byamino acid sequence alignment. TaBZR2 was classi-fied into subgroup V, and the BES/BZRs derived frommonocots clustered separately from those of dicots,suggesting a potential functional diversity betweendicot and monocot plants (Supplemental Fig. S1B).

Drought and Exogenous BR Induced TaBZR2 Expressionand the Nuclear Accumulation of TaBZR2 Protein

We confirmed the expression patterns of TaBZR2 indrought and BR responses by reverse transcriptionquantitative PCR (RT-qPCR) and immunoblot assays.Drought induced TaBZR2 expression in both shootsand roots, reaching a peak at 2 h (Fig. 1, A and B).TaBZR2 expression increased after treatment withexogenous BR and peaked at 4 h in BR-treated leavesand roots (Fig. 1, A and B). Furthermore, drought and

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Functional Analysis of TaBZR2 in Wheat

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exogenous BR treatments increased the abundance ofTaBZR2 protein (Fig. 1, C andD). To better understandthe biological functions of TaBZR2, we investigatedthe subcellular localization of TaBZR2 protein in re-sponse to drought and exogenous BR treatments. TheTaBZR2- GFP fluorescence signal was observed inboth the cytoplasm and nucleus under unstressedconditions (Fig. 1E). Upon drought and exogenous BRtreatments, TaBZR2 proteins translocated from thecytoplasm to the nucleus as shown by the nuclear/cytoplasmic signal ratio (Fig. 1E).

Overexpression of TaBZR2 Significantly ImprovesDrought Tolerance in Transgenic Wheat

To investigate the drought tolerance associated withTaBZR2, we generated transgenic bread wheat plantson the wheat cv ‘Fielder’ background in whichTaBZR2, driven by the maize (Zea mays) Ubiquitinpromoter, was overexpressed. Three independenttransgenic lines that exhibited high TaBZR2 expres-sion level based on RT-qPCR assays were chosen forfurther analysis (Fig. 2B). No differences were ob-served between the TaBZR2-overexpressing (OE5,OE9, and OE11) and wild-type plants under normalgrowth conditions (Fig. 2A). Drought treatmentcaused obvious differences in growth and physiologyof both TaBZR2-overexpressing and wild-type plants.

Upon drought treatment, compared with controlplants, TaBZR2-overexpressing plants had signifi-cantly delayed leaf rolling and higher survival rates(Fig. 2, A and C). Moreover, the Pro contents weresignificantly higher in TaBZR2-overexpressing plantsthan in wild-type plants under drought conditions(Fig. 2D). The TaBZR2-overexpressing plants hadsignificantly lower electrolyte leakage levels andmalondialdehyde (MDA) contents compared to wild-type plants under drought conditions (Fig. 2, E andF). Thus, TaBZR2 regulated physiological processesthat improve the drought tolerance of transgenicwheat plants.

Suppression of TaBZR2 Enhances Drought Sensitivityin Wheat

To further explore the function of TaBZR2 in droughtresponses, we produced two independent TaBZR2-RNAi lines (Ri3 and Ri7) and determined the expres-sion of TaBZR2 using RT-qPCR assays. The expressionlevel of TaBZR2 decreased in the two lines (Fig. 3B;Supplemental Fig. S2), implying that TaBZR2 wassuccessfully suppressed. There were no obvious dif-ferences in the growth performance and physiologybetween TaBZR2-RNAi and wild-type plants undernormal growth conditions (Fig. 3A). However, upondrought treatment, the survival rates were significantly

Figure 1. Expression and localization ofTaBZR2 in wheat under BR and droughtconditions. A and B, The expressionprofile of TaBZR2 in 2-week–old wheatseedling leaf and root tissue underdrought and BR treatments for the indi-cated time. Transcript levels werequantified by RT-qPCR assays. The ex-pression of b-actin was analyzed as in-ternal control. Each data point is themean (6SE) of three experiments. C andD, Protein level of TaBZR2 in 2-week–old wheat seedlings afterdrought and BR treatments for the indi-cated time. Total proteinswere extractedand subjected to immunoblot analysiswith anti-TaBZR2 antibodies. Rubiscowas used as a loading control. E, Lo-calization of TaBZR2 protein underdrought and BR conditions. The nuclear/cytoplasmic signal ratio representsnuclear-accumulated TaBZR2 versuscytoplasmic-accumulated TaBZR2. Im-ages were observed under a laser scan-ning confocal microscope. Scalebar = 12 mm. Each data point is themean (6SE) of 10 biological replicates(**P , 0.01; Student’s t test).

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lower in TaBZR2-RNAi plants than in wild-type plantsunder drought conditions (Fig. 3C). Moreover, drought-treated TaBZR2-RNAi lines had significantly lower Procontents, higher electrolyte leakage levels, and higherMDA contents compared to wild-type plants underdrought conditions (Fig. 3, D–F).

TaBZR2 Positively Regulates the Expression of MultipleStress-Related Genes

To explore how TaBZR2 contributes to drought tol-erance, we performed RNA sequencing (RNA-Seq)assays to evaluate the differential gene expression be-tween TaBZR2-overexpressing and wild-type wheatplants under both normal and drought conditions. Asshown in Figure 4A, using a threshold of a 2-foldchange and a Student’s t test significance cutoff ofP , 0.05, a comparison of the RNA-Seq data fromTaBZR2-overexpressing and wild-type plants undernormal conditions identified 1,399 upregulated and1,064 downregulated genes in TaBZR2-overexpressingplants (TaBZR2-OEN [overexpressing normal]) com-pared with those in wild-type plants (wild-type normal[WTN]). Upon drought treatment, the expression of 728and 1,496 genes in the TaBZR2-overexpressing plants(TaBZR2-OED [overexpressing drought] ) was up- ordownregulated, respectively, compared with that inwild-type plants (wild-type drought [WTD]). In total,20,224 differentially expressed genes (DEGs) wereidentified between drought-treated and normal growth

wild-type plants (WTD/WTN). Cluster and Venn dia-gram analyses revealed that the expression patterns ofall DEGs in TaBZR2-OED compared with that in WTD(TaBZR2-OED/WTD) did not significantly overlapwith TaBZR2-OEN compared with WTN (TaBZR2-OEN/WTN), or WTD compared with WTN (WTD/WTN). These results demonstrated that TaBZR2 sig-nificantly affects the global gene expression profile inwheat, indicating that unknown mechanisms may un-derlie the drought tolerance of transgenic wheat.

Gene ontology (GO) analysis revealed that the DEGsbetween the drought-treated TaBZR2-overexpressingand wild-type plants were significantly enriched in bi-ological process categories including “response toabiotic stimulus,” “response to water stress,” and“regulation of metabolic and biosynthetic processes”(Fig. 4B). Interestingly, we found that the expressions ofa range of well-known stress-related genes were amongthe upregulated DEGs for the drought-treated TaBZR2-overexpressing plants (Supplemental Table S2). Notethat we also used RT-qPCR assays to successfully verifythe upregulated expression trends for the genes iden-tified from the RNA-Seq data, including TaGST1,TaLEA3 (late embryogenesis abundant), TaDHN3(dehydration-stress inducible protein), T. aestivum D-1-pyrroline-5-carboxylate synthetase, TaPOD21 (peroxi-dase), and T. aestivum sucrose non-fermenting1-typeSer/Thr protein kinase (Fig. 4C). Consistent with adirect functional impact of TaBZR2 on the expressionof these known stress-related genes, we also used

Figure 2. TaBZR2-overexpressing wheatplants exhibit improved drought tolerance.A, Phenotypes of TaBZR2-overexpressing(OE5, OE9, and OE11) and wild-typewheat plants under well-watered anddrought conditions. B, RT-qPCR analysisof TaBZR2 gene expression in TaBZR2-overexpressing and wild-type plants. Theexpression of b-actin was analyzed as aninternal control. Each data point is themean (6SE) of three experiments. C, Sur-vival rate of the control and water-stressedplants (without irrigation for 21 d). D, Procontent in seedlings under normal anddrought conditions. E, Electrolyte leakagein seedlings under normal and droughtconditions. F, MDA content in seedlingsunder normal and drought conditions.Each data point is the mean (6SE) of threeexperiments (10 seedlings per experiment).The asterisks indicate significant differ-ences between TaBZR2-overexpressingand wild-type wheat plants (Student’st test, *P , 0.05 and **P , 0.01). WT,wild type.






RT-qPCR to examine the expression of these genes inthe aforementioned drought-treated TaBZR2-RNAiplants and found that their expression was signifi-cantly reduced compared to both drought-treatedTaBZR2-RNAi and wild-type plants (Fig. 4C). GSTgenes, encoding ROS-scavenging enzymes, functionin protecting plants against oxidative damage understress conditions (Jha et al., 2011; Rong et al., 2014). D-1-pyrroline-5-carboxylate synthetase is the key enzymefor Pro synthesis (Yoshiba et al., 1995; Zhuo et al., 2018).Dehydrins are responsive to various environmentalstresses and exhibit multiple biochemical activities,such as buffering water, sequestering ions, stabilizingmembranes, or acting as chaperones (Kovacs et al.,2008; Tang et al., 2012; Rong et al., 2014; Zhuo et al.,2018). Suc non-fermenting-1-related protein kinase2 isimplicated in stress signaling transduction via abscisicacid-dependent and -independent pathways (Yoshidaet al., 2002; Zhang et al., 2011), TaBZR2 could modulatethe expression of numerous stress-responsive genesunder drought conditions, contributing to the droughttolerance of the transgenic wheat.

TaBZR2 Functions Positively in ScavengingDrought-Induced Superoxide Anions

Environmental stimuli, including drought, salt, andhigh and low temperatures, induce the accumulation oftoxic ROS, including H2O2 and superoxide anions(O2

2), which, if not controlled, can eventually lead to

oxidative damage (Dat et al., 2000; Wang et al., 2017).TaBZR2 has a role in activating antioxidant enzymegene expression. To investigate whether TaBZR2 par-ticipates in scavenging ROS, we analyzed the ROScontents between TaBZR2-RNAi and wild-type wheatlines under normal and drought conditions. There wasno significant difference in H2O2 accumulation be-tween TaBZR2-RNAi and wild-type wheat lines underunstressed and drought conditions (Supplemental Fig.S3). The O2

2 contents of TaBZR2-RNAi and wild-typewheat lines were similar under unstressed conditions(Fig. 5, A and B). Nevertheless, the O2

2 contents weresignificantly higher in the TaBZR2-RNAi plants than inthe wild-type plants under drought conditions (Fig. 5, Aand B). 1,3-dimethyl-2-thiourea (DMTU) acts as a O2

2

scavenging reagent (Lv et al., 2018). When DMTU wasadded to the growth medium to reduce O2

2, the O22

contents of TaBZR2-RNAiwheat lines recovered to a levelsimilar to that of the wild-type plants (Fig. 5, A and B).To investigate whether the TaBZR2-mediated O2

2

scavenging was associated with the positive role ofTaBZR2 in drought responses, we compared the bio-mass of TaBZR2-RNAi wheat plants with that of thewild-type wheat plants grown on half-strength Hoag-land’s nutrient solution supplemented with differentconcentrations of polyethylene glycol (PEG) 6000 andDMTU (0, 15% [w/v] PEG 6000, 1 mm of DMTU, and15% [w/v] PEG 6000 + 1 mm of DMTU). Biomass wassimilar for the wild-type and TaBZR2-RNAi plantsgrown on half-strength Hoagland’s nutrient solutioncontaining 0 and 1 mm of DMTU (Fig. 5, C and D).

Figure 3. TaBZR2-RNAi wheat plantsshow enhanced drought sensitivity. A,Phenotypes of TaBZR2-RNAi (Ri3 andRi7) and wild-type wheat plants underwell-watered and drought conditions. B,RT-qPCR analysis of TaBZR2 gene ex-pression in TaBZR2-RNAi and wild-typeplants. The expression of b-actin was an-alyzed as an internal control. Each datapoint is the mean (6SE) of three experi-ments. C, Survival rate of the control andwater-stressed plants (without irrigationfor 18 d). D, Pro content in seedlings un-der normal and drought conditions. E,Electrolyte leakage in seedlings undernormal and drought conditions. F, MDAcontent in seedlings under normal anddrought conditions. Each data point is themean (6SE) of three experiments (10seedlings per experiment). The asterisksindicate significant differences betweenTaBZR2-RNAi and wild-type wheatplants (Student’s t test, *P , 0.05 and**P , 0.01). WT, wild type.


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However, biomass was significantly larger in the wild-type plants than in the TaBZR2-RNAi plants underdrought conditions (Fig. 5, C and D). DMTU treatmentcan partially suppress drought (15% [w/v] PEG 6000)-induced biomass reduction. Importantly, the biomassof TaBZR2-RNAi plants was comparable with that ofthe wild-type plants grown on half-strength Hoag-land’s nutrient solution containing 15% (w/v) PEG6000 and 1mmof DMTU (Fig. 5, C andD). These resultsindicate that TaBZR2 has a role in scavenging O2

2 toalleviate drought stress.

TaBZR2 Functions as a Positive Regulator of TaGST1Expression by Binding to Its Promoter andActivating Transcription

RNA-Seq and RT-qPCR analyses both indicated thatthe expression of TaGST1 is upregulated by TaBZR2

overexpression, so the transcription of this gene may beactivated by TaBZR2. Previous studies have revealedthat BES/BZR family transcription factors can bind toE-box (59-CANNTG-39) cis-elements to regulate theexpression of target genes; we detected 10 E-box cis-elements in the TaGST1 promoter. We thus usedEMSAs to investigate whether TaBZR2 can directlybind to the TaGST1 promoter in vitro. The EMSAsshowed that the TaBZR2-GST fusion protein was ableto bind to the TaGST1 promoter; no such binding wasobserved for the control GST protein (Fig. 6, A and B).Further, the observed binding to the biotin-labeledtarget sequences was dramatically reduced when un-labeled competitor target DNA sequences were added,and no binding was detected when adding the mutatedbiotin-labeled TaGST1 probes (Fig. 6, A and B). Havingdetermined that TaBZR2 can bind the TaGST1 pro-moter in vitro, we next used a wheat protoplast tran-sient expression system to assess whether this binding

Figure 4. Analysis of the expression levels of TaBZR2 downstream genes. A, Venn diagrams comparing the up- and down-regulated genes between wild-type plants under normal and drought conditions (WTN and WTD), and TaBZR2-overexpressingand wild-type plants under normal (TaBZR2-OEN/WTN) and drought conditions (TaBZR2-OED/WTD). B, Functional categori-zation analysis of candidate TaBZR2 target genes in biological process under drought conditions. C, The expression levels ofdrought-responsive genes in TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants. Two-week–old wheat seed-lings treated with 15% (w/v) PEG 6000 for 6 h were used for RNA isolation. Transcript levels were quantified by RT-qPCR assays,and the expression of b-actin was used as an internal control. Each data point is the mean (6SE) of three experiments (10 seedlingsper experiment). WT, wild type.





can drive TaGST1 gene expression in vivo. A pGreen II0800 vector harboring a LUC reporter gene driven bythe TaGST1 promoter (;2,000 bp) was cotransformedinto wheat protoplasts transfected with an emptypJIT16318 vector or a pJIT16318-TaBZR2 vector. Com-pared with the empty-vector control samples, the pro-toplasts expressing TaBZR2 exhibited significantlyincreased expression of the reporter (Fig. 6C). To furthertest if the activation effect of TaBZR2 on the TaGST1wasthrough binding to the E-box (CACGTG, 21,475 to21,481), the TaGST1 promoter containing the mutatedE-box was inserted into the pGreen II 0800 vector andcoexpressedwith the pJIT16318-TaBZR2 vector in wheat

protoplasts. The results demonstrated that the E-boxmutation (AAAAAA, 21,475 to 21,481) disruptedTaBZR2-mediated activation of the TaGST1 promoter(Fig. 6C), indicating that the TaBZR2 transcription fac-tor is a positive regulator of TaGST1 expression.

Overexpression of TaGST1 Significantly ImprovedDrought Tolerance in Transgenic Wheat by ReducingO2

2 Contents

To investigate the function of TaGST1 in the droughtresponse, we generated transgenic wheat plants that

Figure 5. TaBZR2 functions positively in scavenging drought-induced O22. A, NBT staining in primary root tips of TaBZR2-RNAi

andwild-typewheat plants grown in half-strength Hoagland’s liquidmedium,medium containing 15% (w/v) PEG 6000, mediumcontaining 1mM of DMTU, ormedium containing 15% (w/v) PEG 6000 + 1mM of DMTU for 72 h. The strength of color shows theconcentration of O2

2 in the root tips. Scale bar = 1 mm. B, Measurements of the O22 contents of TaBZR2-RNAi and wild-type

wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing1 mM of DMTU, or medium containing 15% (w/v) PEG 6000 + 1 mM of DMTU for 72 h. Each data point is the mean (6SE) of sixbiological replicates. The asterisks indicate significant differences between TaBZR2-RNAi and wild-type wheat plants (Student’st test, **P, 0.01). C, Phenotypes of TaBZR2-RNAi andwild-type wheat plants grown in half-strength Hoagland’s liquidmedium,medium containing 15% PEG 6000, medium containing 1 mM of DMTU, or medium containing 15% (w/v) PEG 6000 + 1 mM ofDMTU. Scale bar = 2 cm. D, Measurement of the total fresh weight of TaBZR2-RNAi and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 1 mM of DMTU, or mediumcontaining 15% (w/v) PEG 6000 + 1 mM of DMTU. Each data point is the mean (6SE) of six biological replicates. The asterisksindicate significant differences between TaBZR2-RNAi and wild-type wheat plants (Student’s t test, *P , 0.05). WT, wild type.


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overexpressed TaGST1 under the control of the maizeUbiquitin promoter. Three independent homozygousT3 transgenic lines with relatively high expression ofTaGST1 were selected for additional phenotypic anal-yses (Fig. 7E). Under normal growth conditions, therewere no notable differences in plant growth or physi-ology between TaGST1-overexpressing (OE1, OE4, andOE9) and wild-type plants. However, upon droughttreatment, the survival rate of TaGST1-overexpressingplants was significantly higher than that of wild-typeplants (Fig. 7, A and B). Moreover, the drought-treatedTaGST1-overexpressing plants had significantly lowerO2

2 content compared to wild-type plants (Fig. 7,C and D).

TaBZR2 Is a Positive Regulator in the BRSignaling Pathway

Toobtainmore detailed evidence for the role ofTaBZR2in BR responses, we transformed the BR-insensitive mu-tant bri1-5with a TaBZR2 overexpression construct underthe control of the Cauliflower mosaic virus (CaMV) 35Spromoter. Two independent homozygous T3 trans-genic lines (35S:TaBZR2/bri1-5-B3 and -B7) thatstrongly expressed TaBZR2 were selected for fur-ther phenotypic analysis. Overexpression ofTaBZR2 partially suppressed the dwarf phenotypesof bri1-5 mutant plants (Supplemental Fig. S4, Aand B). Compared with bri1-5 mutant plants, 35S:

TaBZR2/bri1-5 transgenic plants had enhancedtolerance to the BR biosynthetic inhibitor BRZ(Supplemental Fig. S4B). In addition, comparedwith bri1-5 mutant plants, 35S:TaBZR2/bri1-5transgenic plants showed reduced expression ofthe BR biosynthesis genes Constitutive Photomor-phogenesis and Dwarfism and DWARF4 and in-creased expression of the BR signaling gene thesmall auxin up RNAs (SAUR)-AC (SupplementalFig. S4C).

To obtain further insights into the role of TaBZR2 inthe wheat BR signaling pathway, we investigated theBR sensitivity of TaBZR2 transgenic wheat plants.TaBZR2-overexpressing and TaBZR2-RNAi wheatplants exhibited altered BR sensitivity as indicatedby their root length in the absence or presence of BR. Inthe absence of BR, there was no significant differencein root lengths between TaBZR2-overexpressing,TaBZR2-RNAi, and wild-type wheat plants (Fig. 8A).However, in the presence of BR, the root lengthsof TaBZR2-overexpressing lines were shorter thanthat of wild-type plants. Moreover, compared withwild-type plants, TaBZR2-RNAi plants exhibitedBR-insensitive phenotypes with longer roots (Fig. 8A).Previous studies have shown that BES/BZR familytranscription factors can bind to the BR-response ele-ment (BRRE) in the promoters of feedback-regulatedBR biosynthetic genes to repress their expression (Heet al., 2005). EMSAs demonstrated that TaBZR2 canbind to the BRRE cis-regulatory elements in the

Figure 6. TaBZR2 directly regulates theexpression of TaGST1. A, The diagramshows the structure of the TaGST1 pro-moter. The sequences represent TaGST1probe sequences. The underlined se-quences indicated the core elements ormutated core elements in the TaGST1probe. B, EMSA of TaBZR2 binding to thepromoter of TaGST1. Biotin-labeledTaGST1 probes (normal and mutated)were incubatedwithGSTor GST-TaBZR2protein. 1003 competitor fragmentswere added to analyze the specificity ofbinding. C, TaBZR2 increases TaGST1promoter activity in wheat protoplasts.TaBZR2 was cotransfected with eitherTaGST1 promoter or mutated TaGST1promoter. The LUC to REN ratio is shownand indicates the activity of the tran-scription factors on the expression levelof the promoters. Each data point is themean (6SE) of 10 biological replicates(**P , 0.01; Student’s t test).










promoter of the BR biosynthetic gene T. avestivumCYP90D2 (TaD2; Fig. 8B). Furthermore, RT-qPCR assaysrevealed that, comparedwith wild-type plants, TaBZR2-overexpressing plants showed reduced expression of theBR biosynthetic genes TaD2 and TaDWARF, whereasTaBZR2-RNAi plants showed enhanced expression ofthe BR biosynthetic genes TaD2 and TaDWARF in theabsence and presence of BR (Fig. 8C). The TaBZR2-modulated inhibition of TaD2 and TaDWARF expres-sions were larger in the presence of BR than in theabsence of BR (Fig. 8C). Our data suggest that TaBZR2functions as a positive regulator in BR signaling.

TaBZR2 Is Involved in BR-mediated Drought Responses

To investigate whether TaBZR2 has a role inBR-mediated drought responses, we investigated theexpression of stress-responsive genes encoding anti-oxidant enzymes, including TaGST1, TaPOD21, andTaDHN3, in response to BR treatment by RT-qPCRanalyses. Upon exogenous BR treatment, the expres-sion of these genes in TaBZR2-overexpression plantswas enhanced compared to wild-type plants, whereastheir expression in TaBZR2-RNAi plants was reducedunder normal and drought conditions (Fig. 9A). Inaddition, drought and BR treatments enhanced the

abundance of dephosphorylated TaBZR2 proteins inthe TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type plants (Fig. 9B). The amounts of dephosphorylatedTaBZR2 proteins were larger in TaBZR2-overexpressionplants than in wild-type plants. Nevertheless, comparedto wild-type plants, the amount of dephosphorylatedTaBZR2 proteins in TaBZR2-RNAi plants was smallerupon drought and BR treatments (Fig. 9B). The phos-phorylation status of the BES/BZR family transcriptionfactors is usually used as the biochemical maker forBR signaling outputs (Zhang et al., 2009). Treatmentof immunoprecipitated protein with protein phos-phatase eliminated the slowly migrating band(Supplemental Fig. S5), strongly suggesting that thefast band is unphosphorylated and the slow band isphosphorylated TaBZR2. In addition, the O2

2 accu-mulation of TaBZR2-overexpressing, TaBZR2-RNAi,and wild-type plants was similar under normal condi-tions. When exposed to induced drought conditions,O2

2 accumulation increased in the roots of TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type plants.The O2

2 contents of TaBZR2-overexpressing plants un-der drought conditions was significantly lower than thatof wild-type plants, whereas the O2

2 accumulation wassignificantly higher in TaBZR2-RNAi plants than inwild-type plants (Fig. 9, C and D). Exogenous BR treat-ment repressed the O2

2 accumulation in wheat plants

Figure 7. TaGST1overexpressionpromotesdrought tolerance in transgenic wheat. A,Phenotypes of TaGST1-overexpressing andwild-type plants under normal anddrought conditions. B, Survival rate ofcontrol and water-stressed plants (15%[w/v] PEG 6000 treatment for 14 d).Each data point is the mean (6SE) ofthree experiments (10 seedlings per ex-periment). C, NBT staining in primaryroot tip of TaGST1-overexpressing andwild-type seedlings with 0 or 15% (w/v)PEG 6000 treatment for 72 h. Thestrength of color shows the concentra-tion of O2

2 in the root tips. Scale bar = 1mm. D, Measurements of the O2

2 con-tents of TaGST1-overexpressing and wild-type plants under normal and droughtconditions. Each data point is the mean(6SE) of six biological replicates. E,RT-qPCR analysis of TaGST1 gene ex-pression in TaGST1-overexpressing andwild-typewheat seedlings. The expressionof b-actin was used as an internal control.Each data point is the mean (6SE) ofthree experiments (10 seedlings per experi-ment). The asterisks indicate significant dif-ferences between TaGST1-overexpressingand wild-type plants (Student’s t test, **P,0.01). WT, wild type.


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under normal and drought conditions. Compared towild-type plants, BR-mediated O2

2 scavenging was en-hanced in TaBZR2-overexpressing plants, whereas BR-mediatedO2

2 scavengingwas reduced in TaBZR2-RNAiplants under drought conditions (Fig. 9, C and D). Theseresults indicated that TaBZR2 participates in BR-mediated O2

2 scavenging.

DISCUSSION

Plant genomes have many kinds of transcriptionfactors that function importantly in plant adaption toextreme environmental conditions (Zhang et al., 2017;Liu et al., 2018; Ma et al., 2018). BES/BZR proteinsconstitute another important family of plant-specifictranscription factors (Wang et al., 2002; Yin et al., 2002,2005), but comparatively little is known about their bi-ological functions in drought responses. In this study, adrought-inducible BES/BZR-type transcription factorgene TaBZR2 was identified from wheat drought tran-scriptome data, and follow-up work illustrated thatoverexpression of TaBZR2 enhanced drought tolerancein transgenic wheat plants with larger accumulation of

osmoprotectant metabolites, higher membrane stability,and lower ROS contents compared with the controlplants under drought conditions, whereas TaBZR2-RNAi wheat lines exhibited the opposite trend. Theseresults suggest that TaBZR2 functions positively in reg-ulating drought responses in wheat.

BES/BZR family members regulate the expression oftarget genes by interacting with BRRE and/or or E-boxcis-elements in their promoters (Goda et al., 2004;Nemhauser et al., 2004; He et al., 2005; Wang et al.,2006; Walcher and Nemhauser, 2012; Li et al., 2017).For example, BZR1 binds to BRRE and/or E-box ele-ments in the promoters of the genes encoding CBF1/DREB1A, CBF2/DREB1B, and WRKY6 to modulatetheir expression, contributing to freezing tolerance inArabidopsis (Li et al., 2017), and BES1 directly binds tothe E-box element of the SAUR-AC15 promoter to en-hance auxin signaling in Arabidopsis (Goda et al., 2004;Walcher and Nemhauser, 2012). Our EMSA and LUCreporter analyses demonstrated that TaBZR2 directlybinds to the promoter of TaGST1 to activate its tran-scription. GST genes encode detoxification enzymesthat function in maintaining cell redox homeostasis andprotecting organisms against oxidative stress under

Figure 8. TaBZR2 is a positive regulator in the BR signaling pathway. A, Phenotypes of TaBZR2-overexpressing (OE5, OE9, andOE11), TaBZR2-RNAi (Ri3 and Ri7), and wild-type wheat plants grown in half-strength Hoagland’s liquid medium or mediumcontaining 1 mM of BR. Scale bar = 2 cm. Root length of TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plantsgrown on half-strength Hoagland’s medium that contained different concentrations of BR (0, 0.25, or 1 mM) in the light for 7 d.Each data point is the mean (6SE) of three experiments (20 seedlings per experiment). The asterisks indicate significant differ-ences between TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants (Student’s t test,*P, 0.05). B, EMSA of TaBZR2 binding to the BRREs in the promoter of TaD2. Biotin-labeled BRRE probes (normal andmutated)were incubatedwithGSTorGST-TaBZR2 protein. 1003 competitor fragmentswere added to analyze the specificity of binding. C,The expression levels of BR biosynthetic genes in TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) andwild-type plants. The expression of b-actin was used as an internal control. Each data point is the mean (6SE) of three experiments(10 seedlings per experiment). WT, wild type.





stress conditions (Jha et al., 2011; Rong et al., 2014; Qiet al., 2018). Our data illustrated that, compared withthe wild-type plants, the TaGST1-overexpressingwheatlines exhibited drought tolerance phenotypes withlower O2

2 contents under drought conditions, whichwas consistent with the positive role of TaBZR2 inscavenging drought-induced O2

2. These results indi-cate that TaBZR2 has a role in activating TaGST1 forscavenging O2

2, and consequently alleviate droughtstress.Genetic andmolecular studies have greatly increased

our understanding of the BR signaling pathway in

model plants (Bai et al., 2007; Yu et al., 2008; Ye et al.,2011; Chen et al., 2017). BRs are perceived by aLeucine-rich repeat-receptor kinase, BRI1, andtransduces the signal to activate the BES/BZR familytranscription factor, which regulates the expressionof a large number of genes (Wang et al., 2002; Yinet al., 2002, 2005; Bai et al., 2007; Oh et al., 2012;Jiang et al., 2013; Shimada et al., 2015; Yan et al.,2018). Consistent with the positive role of BES/BZRfamily members in the BR signaling pathway (Wanget al., 2002; Yin et al., 2002; Yan et al., 2018),TaBZR2 positively regulates BR signaling in wheat.

Figure 9. TaBZR2 regulates wheat drought tolerance through the BR-dependent pathway. A, The expression levels of stress-responsive genes in TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants grown inhalf-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 10 nM of BR, or mediumcontaining 15% (w/v) PEG 6000 + 10 nM of BR for 6 h. Each data point is the mean (6SE) of three experiments (10 seedlings perexperiment). B, Protein level of TaBZR2 in TaBZR2-overexpressing, TaBZR2-RNAi, andwild-typewheat plants upon drought andBR treatments for 6 h. Total proteins were extracted and subjected to immunoblot analysis with anti-TaBZR2 antibodies. Rubiscowas used as a loading control. C, NBT staining in primary root tip of TaBZR2-overexpressing, TaBZR2-RNAi, andwild-typewheatplants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 10 nM ofBR, or medium containing 15% (w/v) PEG 6000 + 10 nM of BR for 72 h. The strength of color shows the concentration ofO2

2 in theroot tips. Scale bar = 1mm.D,Measurements of theO2

2 contents of TaBZR2-overexpressing, TaBZR2-RNAi, andwild-typewheatplants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 10 nM ofBR, or medium containing 15% (w/v) PEG 6000 + 10 nM of BR for 72 h. Each data point is the mean (6SE) of six biologicalreplicates. The asterisks indicate significant differences between TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants (Student’s t test, **P , 0.01). WT, wild type.


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Exogenous application of BR protects plants fromdrought stress (Kagale et al., 2007; Xia et al., 2009;Divi et al., 2010, 2016; Nawaz et al., 2017). Previousstudies have shown that some components of the BRsignaling pathway are involved in drought responses(Koh et al., 2007; Sahni et al., 2016). Overexpression ofthe Arabidopsis BR biosynthetic gene DWARF4 con-fers drought tolerance in Brassica napus (Sahni et al.,2016). OsGSK1 is a negative regulator of rice BR sig-naling: its T-DNA knockout mutants display en-hanced tolerance to drought and other abioticstresses (Koh et al., 2007). Considering that TaBZR2functions as a positive regulator in drought re-sponses, it is possible that TaBZR2 has a role in me-diating the crosstalk between BR and droughtresponses. A recent study has shown that BR is in-volved in regulation of the accumulation of O2

2 (Lvet al., 2018). For example, the BR-deficient mutantdet2-9 accumulates more O2

2 in roots (Lv et al., 2018).Our data demonstrated that the expression of someTaBZR2 target genes encoding antioxidant enzymes,including TaGST1, TaPOD21, and TaDHN3, wasupregulated upon exogenous BR treatment. Fur-thermore, exogenous application of BR can enhanceTaBZR2-mediated activation of antioxidant enzymesand scavenging of O2

2 under drought conditions.Our data indicated that TaBZR2 participates in BR-mediated drought response partially by reducing theaccumulation of O2

2.It is worth noting that recent studies also illustrated

that several components of the BR signaling pathwaynegatively regulate drought responses (Chen et al.,2017; Ye et al., 2017). For example, in contrast to apositive role of TaBZR2 in drought responses, AtBES1negatively regulates plant drought tolerance (Ye et al.,2017). BES/BZR family transcription factor genes de-rived frommonocots clustered separately from those ofdicots by phylogenetic analysis. Protein structureanalyses illustrated that the amino acid sequence ofTaBZR2 has an N-terminal binding domain and GSK3-like kinase phosphorylation sites, but no 14-3-3 bindingdomain and PEST motif were identified, which wasdifferent from the well-known BES/BZR family mem-bers like AtBES1, AtBZR1, and OsBZR1 (Wang et al.,2002; Yin et al., 2002; Bai et al., 2007). Furthermore,TaBZR2 exhibited a different BR regulated mobilityshift pattern with AtBZR1 and AtBES1. Previously,studies revealed that all of phosphorylated AtBZR1 andAtBES1 were dephosphorylated upon BR treatment(He et al., 2002; Yin et al., 2002), whereas BR treatmentcaused partially phosphorylated TaBZR2 to convert tothe dephosphorylated form. These results indicate thatalthough BES/BZR familymembers function positivelyin BR signaling, protein structural differences and thedifferent mechanisms of action may lead to functionaldifferences in environmental stress responses. Ourstudy expands the known functional scope of the BES/BZR family members, and its basic insights should in-form the work of both plant abiotic stress researchersand wheat breeders and biotechnologists.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Wheat (Triticum aestivum) plants used for molecular analysis were grown ina greenhouse at 70% relative humidity, 25°C/23°C day/night temperatures,and long-day conditions (16-h light/8-h dark photoperiod) with a light inten-sity of ;300 mmol m22 s21. The wheat cv ‘KeNong 199’ was used to amplifycomplementary DNA (cDNA) sequences of TaBZR2 and TaGST1. The wheat cv‘Fielder’ was used as the receptor material to generate transgenic plants. Toanalyze the expression of TaBZR2 under abiotic stress conditions, wheat cv‘KeNong 199’ seedlings were grown in half-strength Hoagland’s liquid me-dium in a greenhouse with 70% relative humidity, 25°C/23°C day/nighttemperatures, long-day conditions (16-h light/8-h dark photoperiod), with alight intensity of ;400 mmol m22 s21 for 2 weeks. For BR and drought stresstreatments, the roots of wheat seedlings were immersed in half-strengthHoagland’s solution containing 1 mm of 24-epi-brassinolide (EBL) solution(Sigma-Aldrich) and 15% (w/v) PEG 6000. Leaves and roots were sampled at 0,1, 2, 4, 8, 12, and 24 h, and then immediately frozen in liquid N and stored at280°C before RNA extraction. The Arabidopsis (Arabidopsis thaliana) plantswere subsequently grown in a greenhouse at 23°C under long-day conditions(16-h light/8-h dark photoperiod) and a light intensity of ;100 mmol m22 s21.For Arabidopsis, the seeds were germinated on half-strength Murashige andSkoog (Caisson Labs) media supplemented with 2% (w/v) Suc and grown for aweek, after which the seedlings were transplanted into soil. The plants weresubsequently grown in a greenhouse with 70% relative humidity, 23°C, andlong-day conditions (16-h light/8-h dark photoperiod) with a light intensity of;100 mmol m22 s21 for 3 weeks. The Arabidopsis BR-insensitive mutant bri1-5was used for transformation.

Generation of Transgenic Arabidopsis and Wheat

To generate TaBZR2 transgenic wheat plants, the coding regions (codingsequences, CDS) of TaBZR2Dwere cloned into the plant transformation vectorpWMB110 driven by the maize (Zea mays) Ubiquitin promoter. The 198-bpTaBZR2 specific fragment was synthesized by Beijing AuGCT, which wasthen fused in both sense and anti-sense orientations to flank the 508-bp rice(Oryza sativa) zinc finger type family protein gene intron 6. This recombinantDNA was then inserted into the pWMB110 vector to generate the pWMB110-TaBZR2-RNAi construct. To generate TaGST1-overexpressionwheat plants, theTaGST1 CDS were also inserted into the pMWB110 vector, driven by the maizeUbiquitin promoter. Genetic transformations were performed using an Agro-bacterium tumefaciens-mediated transformation system. To isolate positivetransgenic wheat lines, leaves of 10-d–old transgenic wheat seedlings grown inhalf-strength Hoagland’s nutrient solution were used for RNA isolation, andthen RT- and RT-qPCR analyzes were performed. For Arabidopsis, the CDS ofTaBZR2 was introduced into the plant transformation vector pBI121 under thecontrol of the CaMV 35S promoter. The resultant constructs were confirmed bysequencing and then transformed into BR-insensitive mutant bri1-5 plants viathe vacuum infiltration method (Bechtold and Pelletier, 1998). Homozygous T3seeds of the transgenic lines were used for phenotypic analyses. Primers used inthese studies are in Supplemental Table S3.

Drought Stress Treatment

For drought tolerance assays, TaBZR2 transgenic and wild-type wheatseedlings were planted in pots containing mixed soil (1:1 vermiculite/humus)and cultured normally in the greenhouse for 3weeks (until seedlingswere at the3-leaf stage), after which these seedlings were deprived of water until signifi-cant differences in wilting were observed between transgenic and wild-typewheat plants. Three independent experiments were performed. TaGST1transgenic and wild-type wheat seedlings were planted in pots containingmixed soil (1:1 vermiculite/humus) and cultured normally in the greenhousefor 3weeks (until seedlingswere at the 3-leaf stage), after which 15% (w/v) PEG6000 solution was applied to the bottom of the plates for;14 d until significantdifferences in wilting were observed between transgenic and wild-type wheatplants. Three independent experiments were performed.

BR Sensitivity Assays

For BR sensitivity assays, sterilized seeds of TaBZR2-overexpressing,TaBZR2-RNAi, and wild-type wheat plants were maintained at 4°C for 1 week,






after which the germinated seeds were transplanted into half-strength Hoag-land’s solution containing different concentrations of EBL (0, 0.25, and 1 mm).After 7 d of growth at 23°C under long-day conditions (16-h light/8-h darkphotoperiod), images were taken, and the primary root length for each seedlingwas evaluated using an Expression 11000XL Root System Scanning Analyzer(Epson). To perform hypocotyl elongation assays, the sterilized seeds of wild-type plants, 35S:TaBZR2/bri1-5 transgenic Arabidopsis plants, and bri1-5 plantswere sown on half-strengthMurashige and Skoog growthmedia supplementedwith various concentrations (0, 0.25, and 0.5 mm) of BRZ and then kept at 4°C inthe dark for 3 d. After 7 d of growth at 22°C under dark conditions, images weretaken, and the lengths of hypocotyls were measured.

RNA Extraction and RT-qPCR Assays

The total RNA from Arabidopsis and wheat seedlings was extracted usingTrizol reagent (TaKaRa), and their DNAwas digested using RNase-freeDNaseI(TaKaRa). First-strand cDNAwas synthesized using a PrimeScript First-StrandcDNA Synthesis Kit (TaKaRa). RT-qPCRwas performedwith anABI 7500 Real-Time PCR system (Thermo Fisher Scientific) in conjunction with SYBR (ThermoFisher Scientific) to monitor double stranded DNA products. The reaction wasconducted at 95°C for 5min, then 42 cycles of 95°C for 15 s, 58°C to 60°C for 25 s,and 72°C for 30 s. A quantitative analysis using the 2-DDCT method was sub-sequently performed (Le et al., 2011). Each experiment was performed with atleast three independent biological replicates. For each primer pair, the ampli-fication efficiency was checked using a melting-curve analysis. For wheat,b-actin was used as the internal control and actin was used an internal controlfor Arabidopsis (Liu et al., 2013a). The specific primers used for RT-qPCR arelisted in Supplemental Table S3.

Immunoblot Assay

Wheat cv ‘KeNong 199’ seedlings were grown in half-strength Hoagland’sliquid medium in a greenhouse with 70% relative humidity, 25°C/23°C day/night temperatures, and long-day conditions (16-h light/8-h dark photoperiod)with a light intensity of ;400 mmol m22 s21 for 2 weeks. For BR and droughtstress treatments, the roots of wheat seedlings were immersed in half-strengthHoagland’s solution containing 1mmof EBL solution and 15% (w/v) PEG 6000.Leaves were sampled at 0, 2, 4, 8, and 12 h and then used to extract total protein.Plant protein was isolated with lysis buffer (50 mm of Tris at pH 7.5, 1 mm ofEDTA, 150 mm of NaCl, 10 mm of MgCl2, 10% [v/v] glycerol, 1 mm of phe-nylmethanesulfonyl fluoride, 5 mm of dithiothreitol, protease inhibitor cocktailComplete Minitablets [Roche], and 0.2% [v/v] Nonidet P-40). For phosphatasetreatment, the extracted plant proteins were treated with the Lambda proteinphosphatase (P0753S; New England BioLabs) according to the manufacturer’sinstructions. The dephosphorylation reaction took place at 30°C for 30 min in athermal cycler (Bio-Rad). TaBZR2 proteins were subsequently detected byimmunoblotting using Anti-TaBZR2 antibodies at a 1:1,000 dilution. IRDye800CW anti-rabbit IG (H + L) at a 1:10,000 dilution (LI-COR) was used as asecond antibody. The immunoblots were developed via an Odyssey CLx In-frared Imaging System (LI-COR).

Subcellular Localization

Transient expression assayswere conducted as described in Liu et al. (2013a).TaBZR2 was inserted into the subcellular localization vector pJIT16318, whichcontains a CaMV 35S promoter and a C-terminal GFP. Approximately 4 3 104

mesophyll protoplasts were isolated from 10-d–old wheat seedlings and thentransfected with pJIT16318-TaBZR2 plasmids by PEG-mediated transforma-tion. The transfected protoplasts were then incubated at 23°C for 12 h. GFPfluorescence in the transformed protoplasts was imaged using a confocal laser-scanning microscope (LSM 700; Zeiss).

Measurements of Pro Content, Electrolyte Leakage Level,and MDA Content

For assays of physiological traits, 3-week–old wheat seedlings at the 3-leafstage were treated with drought conditions for ;12 d. Approximately 0.2 g ofwheat leaf leaves were harvested for measurements of physiological parame-ters. Absorbance values were measured with a Varioskan LUX MultimodeMicroplate Reader (Thermo Fisher Scientific). The Pro concentration was de-termined as described in Zhang et al. (2012a). The electrolyte leakage was

examined in accordance with methods described in Cao et al. (2007), and theMDA content was assayed as described in Zhang et al. (2012a). All of themeasurements were repeated three times.

Measurements of O22 Content and H2O2 Content

To investigate the contents of O22 and H2O2, 2-week–old wheat seedlings

grown on half-strength Hoagland’s nutrient solution supplemented with dif-ferent concentrations of PEG 6000 and BR (0, 15% [w/v] PEG 6000, 10 nm of BR,15% [w/v] PEG 6000 + 10 nm of BR, 1 mm of DMTU, and 15% [w/v] PEG 6000+ 1 mm of DMTU) for 72 h. The O2

2 contents were measured following theprotocol of the Superoxide Anion Content Detection Kit (BC1295; Solarbio LifeScience). The H2O2 contents were measured following the protocol of the H2O2

Content Detection Kit (BC3595; Solarbio Life Science). For Nitro-blue tetrazo-lium (NBT) staining, the wheat roots were immersed in NBT stain solution for30 min and the dark blue color appeared following the protocol of the AlkalinePhosphatase Activity Detection Kit (Amersco). The staining reaction wasstopped by the addition of an excess of 95% ethanol. Images were observed andphotographed under a stereomicroscope (Leica).

RNA-Seq Assays

TaBZR2-overexpressing (OE9) and wild-type (‘Fielder’) plants were grownin half-strength Hoagland’s liquid medium in a greenhouse with 70% relativehumidity, 25°C/23°C day/night temperatures, and long-day conditions (16-hlight/8-h dark photoperiod) with a light intensity of ;400 mmol m22 s21 for 2weeks. Then, the wheat seedlings were transferred to fresh half-strength Hoag-land’s solution that contained 15% (w/v) PEG 6000. Leaves were sampled at 0and 6 h for transcriptome sequencing experiments, and three biological replicateswere used. The RNA-Seq analysis was performed by the Allwegene Company.Total RNA was extracted from the samples using TRIzol reagent (Invitrogen)according to the manufacturer’s instructions, and RNA sequencing was con-ducted on an IlluminaHiSeq platform. RNA-Seqdatawere analyzed as describedinMortazavi et al. (2008). DEGswere selected using DESeq (1.10.1)with a relativechange threshold of 2-fold (P , 0.05, false discovery rate , 0.01; Anders andHuber, 2010). GO categories were identified using the GOseq R package(Young et al., 2010). The genome annotation and functional categorization arebased on the National Center for Biotechnology Information nonredundantprotein sequences (https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/).

EMSA

The CDS of TaBZR2 was inserted into the pGEX-4T-1 vector. The GST andGST-TaBZR2 fusion proteins were expressed in Escherichia coli (BL21) and pu-rified by glutathione-Sepharose TM 4B (GE Healthcare) according to themanufacturer’s protocol. The biotin-labeled probes used in this assay weresynthesized (Beijing AuGCT), and the sequences are listed in SupplementalTable S3. Double stranded DNA was obtained by heating oligonucleotides at95°C for 10 min and annealing at room temperature. The EMSAwas performedusing the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific)according to the manufacturer’s instructions. In brief, 2 mg of purified fusionprotein GST-TaBZR2 or GST protein was added to the binding reaction. Thebinding reaction took place at 25°C for 30min in a thermal cycler (Bio-Rad). Themixture was separated on a 6% polyacrylamide mini gel, and then the DNAwas transferred to a nylon membrane (Millipore). The signal was visualizedwith an EasySee Western Blot Kit (TransGen).

Transcriptional Activation Assays in Wheat Protoplasts

For the transcriptional activation assay, the promoter fragment of TaGST1was inserted into LUC reporter plasmid pGreen II 0800, which contained aRenilla luciferase (REN) gene under the control of the CaMV 35S promoter usedas an internal control. The effector plasmids and the reporter plasmids werecotransformed into protoplasts by PEG-mediated transformation. After cul-turing for 16 h at 23°C, the activities of LUC and REN were separately deter-mined using a Dual-Luciferase Reporter Assay System (E1910; Promega).

Antibody Preparation

Anti-TaBZR2 was generated by Wuhan Abclonal Biotechnology. TaBZR2CDs (453–999 bp) were inserted into the pET32a vector. Purified His-TaBZR2


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https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/




(151–333 amino acids) fusion protein was injected into rabbits to produceTaBZR2 polyclonal antibodies. Immunoblots were performed using antiserumagainst TaBZR2 and visualized with an EasySee Western Blot Kit (TransGen).

Accession Number

RNA-Seq data described in this study can be found in the National Centerfor Biotechnology Information Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under accession number SRP071191.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Sequence and phylogenetic analyses of TaBZR2.

Supplemental Figure S2. The expression level of BES/BZR family tran-scription factor genes in TaBZR2-RNAi and wild-type wheat plants.

Supplemental Figure S3. Measurements of H2O2 contents in TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants under nor-mal and drought conditions.

Supplemental Figure S4. TaBZR2 overexpression partially rescued thedwarf phenotypes of bri1-5 plants.

Supplemental Figure S5. Immunoblot analysis of TaBZR2 protein.

Supplemental Table S1. Wheat BZRs responsive to drought and BRtreatments.

Supplemental Table S2. Analysis of stress-related genes in TaBZR2-overexpressing and wild-type wheat plants under drought conditions.

Supplemental Table S3. Primers and probes used in this study.

ACKNOWLEDGMENTS

We are grateful to Drs. Rui-Lian Jing and Yong-Fu Fu (Institute of CropScience, Chinese Academy of Agricultural Sciences) for providing wheat seedsand for the BiFC system, respectively. We also thank Dr. Dongying Gao(Department of Plant Sciences, University of Georgia, Athens, GA, USA) forsuggestions on the manuscript.

Received January 28, 2019; accepted February 22, 2019; published March 6,2019.

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