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Plant Physiology and Biochemistry 92 (2015) 92e104

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Molecular characterization of BZR transcription factor family andabiotic stress induced expression profiling in Brassica rapa

Gopal Saha, Jong-In Park, Hee-Jeong Jung, Nasar Uddin Ahmed, Md. Abdul Kayum,Jong-Goo Kang, Ill-Sup Nou*

Department of Horticulture, Sunchon National University, 255 Jungang-ro, Suncheon, Jeonnam 540-950, South Korea

a r t i c l e i n f o

Article history:Received 27 February 2015Received in revised form20 April 2015Accepted 20 April 2015Available online 23 April 2015

Keywords:Abiotic stressBEHBrassica rapaBZRTFs

Abbreviations: BZR, BRASSINAZOLE-RESISTANT;TANT Homolog; TFs, transcription factors; BR, brassBRAD, Brassica database; CDS, coding DNA sequencfunction; bHLH, basic helix-loop-helix; RT-PCR, revechain reaction; qPCR, quantitative polymerase chainmation resource; mL, micro-litre.* Corresponding author.

E-mail address: nis@sunchon.ac.kr (I.-S. Nou).

http://dx.doi.org/10.1016/j.plaphy.2015.04.0130981-9428/© 2015 Published by Elsevier Masson SAS

a b s t r a c t

BRASSINAZOLE-RESISTANT (BZR) transcription factors (TFs) are primarily well known as positive regu-lators of Brassinosteroid (BR) signal transduction in different plants. BR is a plant specific steroid hor-mone, which has multiple stress resistance functions besides various growth regulatory roles. Being animportant regulator of the BR synthesis, BZR TFs might have stress resistance related activities. However,no stress resistance related functional study of BZR TFs has been reported in any crop plants so far.Therefore, this study identified 15 BZR TFs of Brassica rapa (BrBZR) from a genome-wide survey andcharacterized them through sequence analysis and expression profiling against several abiotic stresses.Various systematic in silico analysis of these TFs validated the fundamental properties of BZRs, where ahigh degree of similarity also observed with recognized BZRs of other plant species from the comparisonstudies. In the organ specific expression analyses, 6 BrBZR TFs constitutively expressed in flowerdevelopmental stages indicating their flower specific functions. Subsequently, from the stress resistancerelated expression profiles differential transcript abundance levels were observed by 6 and 11 BrBZRsagainst salt and drought stresses, respectively. All BrBZRs showed several folds up-regulation againstexogenous ABA treatment. All BrBZRs also showed differential expression against low temperature stresstreatments and these TFs were proposed as transcriptional activators of CBF cold response pathway ofB. rapa. Notably, three BrBZRs gave co-responsive expression against all the stresses tested here, sug-gesting their multiple stress resistance related functions. Thus, the findings would be helpful in resolvingthe complex regulatory mechanism of BZRs in stress resistance and further functional genomics study ofthese potential TFs in different Brassica crops.

© 2015 Published by Elsevier Masson SAS.

1. Introduction

BRASSINAZOLE-RESISTANT (BZR) is a group of novel plant-specific transcription factors (TFs) that primarily well known aspositive regulators of Brassinosteroid (BR) signaling process indifferent crops (Li and Deng, 2005; Yin et al., 2005; He et al., 2005).Brassinosteroids (BRs) are a class of plant polyhydroxy steroids thatare involved in the regulation of multiple developmental and

BEH, BRASSINAZOLE-RESIS-inosteroid; Br, Brassica rapa;e; DUF, domain of unknownrse transcription-polymerasereaction; PIR, protein infor-

.

physiological processes including cell elongation, cell division,senescence, vascular differentiation, reproduction, photomorpho-genesis and, responses to various environmental cues (Clouse et al.,1996; Li and Chory, 1997; Ye et al., 2010; Clouse, 2011). Brassinos-teroids (BRs), as stress alleviators dispersed ubiquitously in theplants, protect plants from a variety of environmental stresses,including high- and low-temperature stress, drought, salinity,herbicidal injury, and insect and pathogen attack (Khripach et al.,2000; Bajguz and Hayat, 2009; Campos et al., 2009; Yang et al.,2011). In BR biosynthesis process, the mechanisms and transcrip-tional network through which BZR TFs control BR responses havebeen revealed by identification and characterization of BZR targetgenes in different studies (Yu et al., 2008, 2011; Luo et al., 2010; Sunet al., 2010). Now-a-days, different TF families have been charac-terized as stress responsive besides their growth regulatory func-tions. Being an important factor in the BR synthesis pathway, BZR

G. Saha et al. / Plant Physiology and Biochemistry 92 (2015) 92e104 93

TFs might have stress resistance activities. Surprisingly, no studyregarding stress resistance of BZR TFs in any crop plant has beenreported to date. Therefore, we conducted this study to identify andcharacterize stress regulatory functions of BZR TFs in B. rapa.

BRASSINAZOLE-RESISTANT 1 (BZR1) and BRASSINOSTEROID-INSENSITIVE 1-EMS-SUPPRESSOR 1 (BES1) make up a small genefamily with at least four other members in Arabidopsis, includingBEH1e4. All six members of this plant-specific family likely havepartially redundant functions in BR signaling (Yin et al., 2005; Heet al., 2005). Like BES1 and BZR1, BEH1e4 exist in both phosphor-ylated and dephosphorylated form and accumulate in dephos-phorylated form in response to BR treatment (Yin et al., 2005).Dissection of the functional domains of BZR proteins has revealedhighly conserved N-terminal domains that have DNA binding ac-tivity both in vitro and in vivo (Yin et al., 2005; He et al., 2005). TheBZR1 DNA binding domain (encoded by the first exon) is the mostconserved region of the BZR1-related proteins and is thus namedthe BZR domain (He et al., 2005). BZR1 and BES1/BZR2 are twowell-characterized transcription factors in the BR signalingpathway. These transcription factors are unique to plants and sharehigh similarity at the amino acid level (Wang et al., 2002; Yin et al.,2002; Zhao et al., 2002). Both BZR1 and BES1 have an atypical basichelix-loop-helix (bHLH) DNA binding motif in the N terminus andbind to E-box (CANNTG) and BRRE (CGTGT/CG) elements, respec-tively (He et al., 2005; Yin et al., 2005). The central regions of theseproteins contain 22e24 putative phosphorylation sites for theGSK3 kinase family, and several contain a putative PEST motifinvolved in protein degradation (Yin et al., 2005).

The genus Brassica includes a number of important crops thatserve as a source of oil, vegetables, condiments, dietary fiber, andvitamin C (Talalay and Fahey, 2001). Among the species in thisgenus, B. rapa comprises several important subspecies, such asChinese cabbage (B. rapa ssp. pekinensis), non-heading Chinesecabbage (B. rapa ssp. chinensis), and turnip (B. rapa ssp. rapifera).Chinese cabbage is one of the most important vegetables in Asia.B. rapa is the model species representing the Brassica ‘A’ genomeand was therefore selected for genome sequencing (BrassicaGenome Gateway: http://brassica.bbsrc.ac.uk; The Korea Brassicarapa Genome Project: http://www.brassica-rapa.org/BRGP/index.jsp). Comparative genomic analysis confirmed that B. rapa under-went genome triplication since its divergence from Arabidopsis(Song et al., 2013). This species has already proven to be a usefulmodel for studying polyploidy because it has a relatively smallgenome (approximately 529 megabase-pairs [Mbp]) comparedwith other Brassica species. Numerous studies have focused on theregulatory roles of BZR TFs in BR signal transduction in Arabidopsis,a close relative of B. rapa. However, the stress responsiveness of BZRTFs in any crop species remains to be investigated. In this study, weselected B. rapa as an important vegetable crop worldwide that issubjected to a variety of abiotic stresses to investigate the stressresponsiveness of BrBZR TFs.

The recent sequencing of the B. rapa ssp. pekinensis genome(Wang et al., 2011) offers the possibility of genome-wide analysis ofBZR TFs. In this study, we analyzed the genomic localizations,protein motif structures, phylogenetic relationships, and structuresof candidate BZR TFs in B. rapa. In addition, we carried out genome-wide expression profiling of these genes in plants subjected to low-temperature treatment involving the cold-tolerant and -susceptibleinbred B. rapa lines ‘Chiifu’ and ‘Kenshin’, respectively. We furtherinvestigated the expression patterns of these TFs in response to salt,drought and ABA treatment and examined their tissue-specificexpression in different organs. We observed constitutive expres-sion of several BrBZR TFs throughout the flower bud developmentstages. We identified BrBZR TFs with co-responsive expressionsagainst stress treatments, suggesting that they play multiple roles

in stress resistance in B. rapa.

2. Materials and methods

2.1. Identification of BZR TFs

A search on SWISSPROT of the Brassica database (BRAD) wasconducted using keyword ‘BZR’ (http://brassicadb.org/brad/; Chenget al., 2011). Protein sequences and CDS of the identified B. rapa BZRTFs were obtained from the Brassica database (http://brassicadb.org/brad/; Cheng et al., 2011). The web tool ‘SMART’ from EMBL(http://smart.embl.de/smart/set_mode.cgi?GENOMIC¼1) was usedto confirm the identity of the BZR domain, and a protein homologystudy was conducted using the Basic Local Alignment Search Tool(BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm the pu-tative BZR TFs in B. rapa. The primary structures of the genes wereanalyzed using protParam (http://expasy.org/tools/protparam.html). Multiple protein sequences were aligned using PIR (http://pir.georgetown.edu/pirwww/search/multialn.shtml).The numberof introns and exons was determined by aligning the CDS with thegenomic sequences using the Gene Structure Display Server (GSDS)web tool (Guo et al., 2007). The subcellular localization of BrBZRproteins was predicted using ProtComp 9.0 from Softberry (http://linux1.softberry.com/berry.phtml). To identify the putative cis-acting regulatory elements of BrBZR TFs, about 1500-bp CDS se-quences from the translation initiation codon (ATG) was analyzedusing PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

2.2. Phylogenetic analysis of BZR TFs

B. rapa BZR TF sequences were aligned with those of rice andArabidopsis using ClustalX (Thompson et al., 1997). Phylogenetictrees were generated with MEGA6.06 using the Neighbor-Joining(NJ) algorithm (Tamura et al., 2013). Bootstrap analysis with 1000replicates was used to evaluate the significance of the nodes.Pairwise gap deletion mode was used to ensure that the divergentdomains could contribute to the topology of the NJ tree.

2.3. Analysis of conserved motifs in BZR TFs

The BZR protein sequences were analyzed usingMEME software(Multiple Em for Motif Elicitation, V4.9.0) (Bailey et al., 2006). AMEME search was executed with the following parameters: (1)optimum motif width �6 and �200; (2) maximum number ofmotifs to identify ¼ 10.

2.4. Chromosomal locations and gene duplication of BZR TFs

All BZR TFs of B. rapa were BLAST searched (http://www.ncbi.nlm.nih.gov/BLAST/) against each other to identify duplicategenes, where both the similarity and query coverage percentage ofthe candidate genes were >80% (Kong et al., 2013). Positional in-formation about all candidate BZR TFs along the 10 chromosomes ofB. rapawas obtained from the Brassica database (http://brassicadb.org/brad/; Cheng et al., 2011). The positions of the genes along 10chromosomes and duplication lines among genes on the chromo-somes were drawn manually.

2.5. Collection and preparation of plant material

B. rapa ‘SUN-3061’ plants were grown at the Department ofHorticulture, Sunchon National University, Korea. For the organstudy, fresh roots, stems, leaves, and flower buds were harvested,frozen immediately in liquid nitrogen, and stored at�80 �C for RNA

G. Saha et al. / Plant Physiology and Biochemistry 92 (2015) 92e10494

isolation. For the three abiotic stress treatment, two inbred lines ofB. rapa ssp. pekinensis, i.e., ‘Chiifu’ and ‘Kenshin’, were used. ‘Chiifu’originated in temperate regions, whereas ‘Kenshin’ originated insubtropical and tropical regions (Lee et al., 2013). Plants werecultivated under aseptic conditions in semi-solid medium for fourweeks. Three stress treatments, including cold, drought, and salt,were administered for various durations (0 h, 1 h, 4 h,12 h, 24 h and48 h). Plant samples were transferred to an incubator at 4 �C toinduce cold stress. Drought/desiccation stress was simulated bydrying the plants on Whatmann 3 mm filter sheets. To induce saltand ABA stress, plant samples were transferred to rectangular Petridishes (72 � 72 � 100 mm) containing 200 mM NaCl and 100 mMabscisic acid (ABA) for the designated time period, respectively(Arora et al., 2007; Chen et al., 2006). For each stress treatment,leaves of treated samples were collected and processed for analysisof the expression patterns of different BZR TFs.

2.6. RT-PCR expression analysis

RT-PCR was conducted using an AMV one step RT-PCR kit(Takara, Japan). Specific primers for all genes were used for RT-PCR,and Actin primers from B. rapa (FJ969844) were used as a control(Supplementary Table 3). PCR was conducted using 50 ng cDNAfrom the plant and flower organs as templates in master mixescomposed of 20 pmol each primer, 150 mM each dNTP, 1.2 U Taqpolymerase, 1� Taq polymerase buffer, and double-distilled H2O ina total volume of 20 mL in 0.5-mL PCR tubes. The samples weresubjected to the following conditions: pre-denaturing at 94 �C for5 min, followed by 30 cycles of denaturation at 94 �C for 30 s,annealing at 55 �C for 30 s, and extension at 72 �C for 45 s, with afinal extension for 5 min at 72 �C.

2.7. Quantitative PCR expression analysis

Real-time quantitative PCR was performed using 1 mL cDNA in a10-mL reaction volume employing iTaqTM SYBR® Green Super-mixwith ROX (California, USA). The specific primers used for real-timePCR are listed in Supplementary Table 3. The conditions for real-time PCR were as follows: 5 min at 95 �C, followed by 40 cyclesat 95 �C for 10 s, 55 �C for 10 s, and 72 �C for 15 s. The fluorescencewas measured following the last step of each cycle, and threereplications were used per sample. Amplification detection anddata analysis were conducted using LightCycler96 (Roche,Germany).

Table 1In silico analysis of BZR genes collected from the Brassica database.a

Gene name Locus ID Located chromosomenumber

ORF (bp) Protein

Length (aa)

BrBZR1-1 Bra003772 A07 996 331BrBZR1-2 Bra008378 A02 945 314BrBES1-1 Bra010093 A06 1002 333BrBES1-2 Bra011706 A01 1015 331BrBEH1-3 Bra008177 A02 1161 386BrBEH1 Bra012570 A03 879 292BrBEH2 Bra012891 A03 924 307BrBEH3 Bra013359 A01 675 224BrBEH4 Bra015868 A07 831 276BrBEH5 Bra016508 A08 831 276BrBEH6 Bra017782 A03 459 152BrBEH7 Bra020986 A08 912 303BrBEH8 Bra025733 A06 942 313BrBEH9 Bra031077 A09 857 285BrBEH10 Bra035060 A07 636 211

Abbreviations: ORF, open reading frame; bp, base pair; aa, amino acid; kDa, kilo Dalton;a Brassica database (http://brassica.org/brad/index.php).

3. Results

3.1. Sequence analysis and phylogenetic classification of BZR TFs inB. rapa

We identified 15 BZR TFs in B. rapa, which we designated B. rapaBZR (BrBZR) TFs. The predicted sizes of the 15 BrBZRs ranged from152 to 386 amino acids (16.46e42.53 kDa), and the predicted iso-electric points varied from 4.44 to 9.48 (Table 1). Analysis of theprotein domain organization showed that 13 BrBZR TFs containedthe ‘DUF822’ domain, which is characteristic of BZR TFs. Theremaining two proteins exhibited other distinguishing BZR TFsfeatures but lacked the ‘DUF822’ domain. Additionally, the DNAsequences of the 15 BrBZR TFs were determined based on theB. rapa whole-genome sequence. Analysis of the intron and exondistribution showed that most of the genes exhibited similarsplicing patterns (Fig. 1). A BLAST search of the NCBI database tocompare the 15 BrBZRs with BZRs of other species revealed that thededuced amino acid sequences of all 15 BrBZRs shared the highestsimilarity levels with Arabidopsis BZR TFs. The sequence similarityranged from 50 to 94%, more specifically, 12 BrBZRs shared greaterthan 80% similarity with Arabidopsis BZR TFs (Table 2). The simi-larity among the BrBZR TFs sequences ranged from 57 to 96%, and11 BrBZRs shared greater than 80% similarity within the species,indicating their probable duplication (Fig. 2).

We also retrieved the sequences of all Arabidopsis and rice BZRTFs fromNCBI and constructed a phylogenetic treewith 15 deducedamino acid sequences of BrBZR using the NJ method (Fig. 3). In thephylogenetic tree, we also identified close homologs of ArabidopsisBZR and BZR-homolog (BEH). Two BrBZR TFs formed a tight groupwith BZR1, which we subsequently designated BrBZR1-1 andBrBZR1-2. Accordingly, three TFs closely related to BZR2/BES1 weredesignated BrBES1-1, BrBES1-2, and BrBES1-3. The remaining 10BZR TFs were closely grouped with Arabidopsis BZR1-homologs(BEH) and were therefore designated BrBEH1e10. All of the riceBZR TFs exhibited distant relationships with both B. rapa and Ara-bidopsis BZR TFs, especially with the BZR1 and BZR2/BES1 cluster inthe phylogenetic tree. This result suggests that expansion of BZR1and BZR2/BES1 took place after the divergence of dicots andmonocots.

Motif searching identified 10 motifs which are characteristic toBrBZR TFs (Fig. 1 and Supplementary Table 1). Motif searching andmultiple sequence alignments revealed thatmost of the TFs have anatypical basic helix-loop-helix (bHLH) DNA-bindingmotif in their N

Sub-cellularlocalization

No. ofintrons

BZR domain starteend (aa) PI MW (kDa)

17e170 9.05 35.87 N 116e160 9.40 34.34 N 116e171 9.26 36.17 N 117e164 9.44 36.18 N 181e223 9.41 42.53 N 22e153 8.72 31.41 N 12e155 7.57 33.06 N 1None 4.99 24.04 EC 22e141 8.52 30.03 N 12e141 8.69 30.05 N 1None 4.44 16.46 EC 09e152 8.63 33.09 N 114e169 9.16 33.73 N 115e166 9.48 30.74 N 17e111 5.43 22.41 N 1

PI, iso-electric point; MW, molecular weight; N, nuclear; EC, extracellular.

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G. Saha et al. / Plant Physiology and Biochemistry 92 (2015) 92e104 95

termini represented by motif 1 (Figs. 1 and 4). The middle portionsof these TFs harbor serine (S)-rich phosphorylation sites and a PESTdomain, which are represented bymotif 6 andmotif 5, respectively.PEST domains are involved in controlling protein stability (Yin et al.,2005). Motif 2 and 4 are characteristic of the C-terminal domain.Multiple sequence alignments revealed this region to be highlyconserved among all of the proteins. Although BrBEH3 and BrBEH6lackmotif 1, they possess four other characteristic motifs of the BZRfamily. Among the 15 BrBZR TFs, 12 contain a single intron withsimilar splicing patterns (‘0’), while the splicing pattern of BrBEH10is ‘2’. Among the remaining three TFs (BrBES1-3, BrBEH3, andBrBEH6), BrBES1-3 and BrBEH3 have double introns, while BrBEH6lacks any introns (Fig. 1).

We predicted subcellular localization of the 15 BrBZR TFs usingProtcomp 9.0 from Softberry. As transcription factor family pro-teins, all but two BrBZRs were identified to have nuclear localiza-tion (Table 1). The exception is in case of BrBEH3 and BrBEH6 andtheywere found to be located as extracellular proteins of B. rapa. Toidentify the putative cis-acting regulatory elements in BrBZR TFs,about 1500-bp CDS sequences from the start codons (ATG) werecritically examined. The identified cis-elements included abioticstress responsive elements, as well as those involved in light andcircadian rhythm regulation, disease resistance and seed develop-ment, such as: TC-rich repeats, TCA element, HSE element, EREelement, Box-W1 element, MBS element, LTR element, CGTCAmotif, TGACG-motif, TGA element, ABRE, CE3, motif IIb, GARE-motif, P-box, SARE, AuxRR-core, GC-motif, WUN-motif and circa-dian (Supplementary Table 2).

3.2. Organ-specific expression analysis under unstressed conditions

We performed RT-PCR analysis using specific primers withequal amounts of cDNA templates prepared from the mRNA ofroots, stems, leaves, and flower buds of healthy, unstressed Chinesecabbage plants to examine the organ-specific expression patternsof the BrBZR TFs. BrBZR1-1, BrBZR1-2, BrBES1-1, BrBES1-2, BrBEH1,and BrBEH4e7were ubiquitously expressed in all organs examined,with variable transcript levels (Fig. 5a). Specifically, all TFs exceptBrBEH2 were expressed in the flower buds of B. rapa. BrBEH2 wasnot expressed in any of the tissues examined. We further investi-gated the expression of all BrBZR TFs in flower buds at six stages ofdevelopment (fromvery young tomature buds). All TFs had distinctexpression patterns at all developmental stages. Intriguingly, theexpression of BrBES1-1, BrBEH1, BrBEH3, BrBEH7, and BrBEH8gradually increased until the mature bud stage (Fig. 5b). Bycontrast, the expression of BrBEH2 gradually increased to devel-opmental stage four (S4) and decreased gradually in stages five andsix. These six TFs may play regulatory roles in flower bud devel-opment in B. rapa.

3.3. Expression analysis under abiotic stress conditions

3.3.1. Microarray analysis of gene expression in response to coldand freezing stress

We analyzed microarray expressions of 15 BrBZRs from ourpreviously published data set, where two contrasting inbred linesof B. rapa, Chiifu and Kenshin were treated with cold and freezingstresses (4 �C, 0 �C, �2 �C and �4 �C) for 2 h (Jung et al., 2014)(Fig. 6). Chiifu and Kenshin, have different geographic origins;Chiifu originated in temperate regions, whereas Kenshin originatedin subtropical and tropical regions. Therefore, they are expected torespond differently to cold and freezing temperatures. In nature,Kenshin exhibits severe damage upon exposure to low tempera-tures, while Chiifu does not exhibit such damage (Dong et al., 2014).Moreover, Kenshin has been used as a breeding stock to develop

Table 2Homology analysis of 15 BZR proteins in B. rapa.

Sl. Gene name Top matched clones Name of protein % Identity E-value Top homologous species Refs.

1. BrBZR1-1 NP_565099 Brassinazole-resistant 1 protein 92% 0 A. thaliana Theologis et al., 20002. BrBZR1-2 NP_565099 Brassinazole-resistant 1 protein 81% 5.00E-161 A. thaliana Theologis et al., 20003. BrBES1-1 NP_973863 Protein brassinazole-resistant 2 86% 1.00E-167 A. thaliana Theologis et al., 20004. BrBES1-2 NP_973863 Protein brassinazole-resistant 2 88% 9.00E-177 A. thaliana Theologis et al., 20005. BrBES1-3 NP_973863 Protein brassinazole-resistant 2 78% 5.00E-163 A. thaliana Theologis et al., 20006. BrBEH1 NP_565187 BES1/BZR1 homolog protein 4 86% 2.00E-152 A. thaliana Theologis et al., 20007. BrBEH2 NP_565187 BES1/BZR1 homolog protein 4 84% 4.00E-155 A. thaliana Theologis et al., 20008. BrBEH3 NP_565187 BES1/BZR1 homolog protein 4 63% 1.00E-36 A. thaliana Theologis et al., 20009. BrBEH4 NP_193624 BES1/BZR1 homolog protein 3 93% 8.00E-164 A. thaliana Mayer et al., 199910. BrBEH5 NP_193624 BES1/BZR1 homolog protein 3 93% 4.00E-165 A. thaliana Mayer et al., 199911. BrBEH6 NP_193624 BES1/BZR1 homolog protein 3 94% 2.00E-75 A. thaliana Mayer et al., 199912. BrBEH7 Q94A43 BES1/BZR1 homolog protein 2 91% 2.00E-167 A. thaliana Bevan et al., 199813. BrBEH8 Q94A43 BES1/BZR1 homolog protein 2 90% 2.00E-166 A. thaliana Bevan et al., 199814. BrBEH9 NP_190644 BES1/BZR1 homolog protein 1 87% 6.00E-140 A. thaliana Salanoubat et al., 200015. BrBEH10 NP_565099 brassinazole-resistant 1 protein 50% 3.00E-21 A. thaliana Theologis et al., 2000

A10A01 A02 A03 A04 A05 A06 A07 A08 A09

BrBES1-2 (+)

BrBEH3 (-)

BrBZR1-2 (+)

BrBES1-3 (-)

BrBEH1 (+)

BrBEH2 (+)

BrBEH6 (+)

BrBES1-1(-)

BrBEH8 (-)

BrBZR1-1(-)

BrBEH4 (+)

BrBEH10 (+)

BrBEH5 (+)

BrBEH7 (+)

BrBEH9(-)

Fig. 2. Chromosomal locations of B. rapa BZR TFs along ten chromosomes. Chromosome numbers A01 to A10 are indicated above each chromosome. Different colors of gene namesrepresent different types of BZRs (black: BrBEH, blue: BrBES, and red: BrBZR). The plus (þ) and minus (�) signs following each gene name represent forward and reverse orientationof the respective gene. Genes on duplicated segments of the genome are joined by black dotted lines. Gene positions and chromosome sizes can be estimated using the scale (inMegabases; Mb) to the left of the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

G. Saha et al. / Plant Physiology and Biochemistry 92 (2015) 92e10496

heat tolerant plants (Yamagishi et al., 1994).We observed that BrBZR1-1, BrBES1-3, BrBEH4, BrBEH6, BrBEH7,

and BrBEH8 gave differential cold- or freezing-responsive expres-sion patterns between the two lines, while the remaining genesexhibited little or no expression. BrBEH4 was differentiallyexpressed between the two lines. BrBEH6, BrBEH7, and BrBEH8exhibited variable expression patterns at different temperatures in“Chiifu”. Moreover, in “Chiifu”, BrBZR1-1 and BrBES1-3 were morehighly expressed in response to cold (0 and 4 �C) versus freezingtemperatures. By contrast, in ‘Kenshin’, BrBEH8 was highlyexpressed upon exposure to both cold and freezing temperatures.In summary, the BrBEH TFs were more highly induced than theBrBZR1 and BrBES1 TFs in response to cold or freezing stress.

3.3.2. Quantitative PCR analysis of gene expression under abioticstress conditions

One of our main objectives was to identify BrBZR TFs that arestress responsive in addition to playing various roles in plantgrowth. To further elucidate (and to validate) the responses of the

BrBZR TFs against cold stress, we used two inbred lines of B. rapa,Chiifu and Kenshin for the qPCR experiment. In addition to coldstress, we also examined the salt, drought, and ABA responsivenessof all BZR TFs in B. rapa ‘Chiifu’ (Fig. 7aed).

In ‘Chiifu’, BrBZR1-1, BrBZR1-2, BrBES1-2, BrBEH4, BrBEH6,BrBEH8, BrBEH9, and BrBEH10 were differentially expressed inresponse to cold stress; the expression of these TFs graduallyincreased from 0 h to 12 h and immediately decreased at 24 h.Among these TFs, only BrBEH4 and BrBEH6 were up-regulated at48 h BrBEH1, BrBEH2, BrBEH3, and BrBEH5 were up-regulated at12 h and were expressed at (approximately) control levels at theother time points. BrBEH7 was down-regulated from the verybeginning of stress treatment. Conversely, in ‘Kenshin’, most ofthese TFs did not show any significant changes in expression, orwere down-regulated in response to cold stress (Fig. 7a).

In B. rapa ‘Chiifu’, BrBES1-2, BrBH4, BrBEH5, BrBEH6, BrBEH7,BrBEH8, and BrBEH10 were considerably up-regulated in responseto salt treatment until the middle stage of treatment, followed bydown-regulation to minimum levels, which were maintained

Fig. 3. Phylogenetic tree showing the relatedness of the deduced full-length aminoacid sequences of 15 BrBZR proteins and all BZR family proteins of Arabidopsis and rice.BrBZR proteins are shown in blue. The scale represents the frequency of amino acidsubstitutions between sequences as determined by the Poisson evolutionary distancemethod. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

G. Saha et al. / Plant Physiology and Biochemistry 92 (2015) 92e104 97

through the end of treatment. By contrast, BrBES1-3 and BrBEH9were down-regulated from the beginning of stress treatment,becoming inactive at 48 h. The expression levels of the remainingTFs nearly matched those of the control (Fig. 7b).

We identified some BrBZR TFs as candidates that might beinvolved in resistance against drought stress in B. rapa ‘Chiifu’.Notably, BrBES1-1, BrBES1-2, BrBEH1, BrBEH3, BrBEH4, BrBEH5,BrBEH6, BrBEH7, BrBEH8, and BrBEH10 were up-regulated in boththe early and late hours of stress treatment. Conversely, the down-regulation of BrBES1-3 and BrBEH9 throughout the stress period isalso notable. These TFs became inactive in the final hour of stresstreatment; their susceptibility to drought stress might have someconnection with the stress resistance of B. rapa ‘Chiifu’ (Fig. 7c).

Most of the BrBZR TFs in B. rapa ‘Chiifu’ were up-regulated inresponse to ABA treatment. Specifically, BrBZR1-1 and BrBES1-2were gradually up-regulated until 48 h of ABA treatment.

BrBZR1-2 and BrBES1-3 were down-regulated during the earlystage of hormone treatment, which was followed by up-regulationuntil 48 h of treatment. The remaining BrBZR TFs were up- ordown-regulated up to the last stage of ABA treatment, except forBrBEH2, which was down-regulated during the later hours oftreatment and eventually became inactive (Fig. 7d).

Our results reveal that more BrBZR TFs were up-regulated in‘Chiifu’ (a cold-resistant line) than in ‘Kenshin’ in response to coldstress. The highly expressed BrBZR TFs in ‘Chiifu’might be involvedin cold resistance, while their inactivity or very low activity in‘Kenshin’might play a role in the cold susceptibility of this line. Wealso identified some candidate BrBZR TFs that showed considerableresponsiveness against salt, drought, and ABA stress in B. rapa. Wefound that BrBEH4, BrBEH5 and BrBEH6 exhibited similar expres-sion patterns in response to all four stresses, suggesting that theseTFs might have multiple stress resistance-related functions inB. rapa.

3.4. Analysis of BrBZR protein interactions

We investigated the interactions of the 15 B. rapa BZR TFs andtheir association with Arabidopsis proteins, including their func-tional and physical interactions, using STRING software (Fig. 8).BrBZR1-1, BrBZR1-2, BrBEH10, BrBES1-1, BrBES1-2, and BrBES1-3,which are highly homologous to BRASSINAZOLE-RESISTANT 1(BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) proteins of Arabi-dopsis, respectively, are involved in a brassinosteroid (BR) signalingnetwork. BRASSINOSTEROID-INSENSITIVE 2 (BIN2), BRASSINOS-TEROID INSENSITIVE 1, BRI1 SUPPRESSOR 1 (BSU1), EARLY FLOW-ERING 6 (ELF6), RELATIVE OF EARLY FLOWERING 6 (REF6),GENERAL REGULATORY FACTOR 8 (GRF8), GENERAL REGULATORYFACTOR 10 (GRF10), DWARF 4 (DWF4), BES1-INTERACTING MYC-LIKE 1 (BIM1), and HIGH NITROGEN INSENSITIVE 9 (HNI9) appearto participate in this process. The six above-mentioned B. rapa BZRTFs have evolved as functional partners in this interaction network.We found that these five B. rapa BZR TFs are responsive to ABA andare strongly associated with Arabidopsis GRF10 and DWF4, whichare involved in ABA and jasmonic acid resistance, respectively (Renet al., 2009; Kim et al., 2013). The 10 remaining B. rapa BZR homologTFs (BrBEH1e9) are highly homologous to four unknown Arabi-dopsis proteins (AT4G18890, AT4G36780, AT1G78700, andAT3G50750), which might play positive regulatory roles in brassi-nosteroid signaling, like BZR1; Yin et al. (2005) reported their ge-netic redundancy (identified as BZR homologs) in BR regulation.The tissue-specific and stress-responsive expression patterns ofthese TFs were similar to those of BrBZR1 and BrBES1.

4. Discussion

The BZR TF family appears to be involved in the regulation ofvarious processes in plants. While major studies have revealed thepositive roles of these TFs in BR signal transduction (He et al., 2005;Yin et al., 2005), no genome-wide, in-depth study of the BZR TFfamily has previously been reported. Complete and accurateannotation of genes/proteins is an essential starting point forfunctional studies of transcription factor families. In our currentgenome-wide study of B. rapa, we investigated the organ-specificexpression of 15 BrBZR TFs; their ubiquitous expression in mostorgans suggests that they might function as growth regulatorsduring B. rapa development. A more thorough investigationrevealed that these genes were differentially expressed duringflower bud development (Fig. 5b). The function of these TFs in plantorgan growth and development has not previously been reported.The expression patterns of these TFs during flower bud develop-ment makes them good candidates for further investigations of

Fig. 4. Alignment of deduced amino acid sequences of BrBZR proteins using PIR. Amino acids shaded with different colors are conserved. Solid boxes represent N- and C-terminal DUF822 and C-terminal domains, respectively. Blue linesabove the DUF822 domain indicate the basic helix-loop-helix-like structure. Red lines indicate serine (S)-rich phosphorylation sites and ‘PEST’ sequences among the 15 BrBZR proteins. Numbers at the top indicate the positions of aminoacid residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. RT-PCR expression of 15 BZR TFs in different tissues of B. rapa are showing in (a) roots, stems, leaves, and flower buds (lane 1 to 4, respectively) and (b) six floral growthstages (young to mature buds are marked S1 to S6 at the top of the figure). Transcript accumulations were recorded at 30 cycles, except in the cases of BrBES1-1, BrBEH1 and BrBEH2(35 cycles).

Fig. 6. Microarray expression analysis of 15 BZR TFs in B. rapa under different temperature treatments. C and K indicate inbred B. rapa lines ‘Chiifu’ and ‘Kenshin’, respectively,treated under five different temperature conditions: control (C1 and K1), 4 �C (C2 and K2), 0 �C (C3 and K3), �2 �C (C4 and K4), and �4 �C (C5 and K5). Color bar at the base indicatesexpression levels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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their growth-related functions. More importantly, in the studyagainst cold, salt, drought, and ABA stresses, we found differentialexpression patterns of all the BrBZR TFs. Notably, all 15 BrBZR TFscontained predicted abiotic stress responsive cis-acting elements intheir sequences. Outcomes of our study might be the foundationwork to undertake further functional studies on these potential BZRTFs, which ultimately can provide a wider basis for exploitation ofthese candidate genes for genetic engineering of B. rapa.

Most of the BrBZR TFs were more highly up-regulated (byseveral fold) in response to cold stress in the cold-resistant line(‘Chiifu’) than in the cold-susceptible line (‘Kenshin’), suggestingthat these genes might play a role in the cold resistance of B. rapa‘Chiifu’. To uncover the cold resistance mechanism of BrBZR TFfamily, we investigated their sequence organization and foundconserved atypical bHLH domain in the N-terminal region which

also has been reported by He et al. (2005). Moreover, their cis-element analysis in all the gene sequences revealed E-box bindingelement (CANNTG) which is designated as ‘MBS’ (MYB bindingsites) (Supplementary Table 2). We speculate our candidate BrBZRTFs also respond against cold/freezing binding with the coreelement (CANNTG) of C-REPEAT BINDING FACTOR (CBF) and regulatecold responsive genes [COLD REGULATING (COR) GENE] in thedownstream to increase cold/freezing tolerance of B. rapa. Jianget al. (2011) in their study presented evidence for a CBF cold-response pathway in non-heading Chinese cabbage (Brassica cam-pestris ssp. chinensis L Makino). They showed, BrICE1, a bHLH typetranscription factor, which regulates CBF-cold responsive pathway.In this study, we also want to propose that our candidate BrBZR TFshaving atypical bHLH domain organization those showed resis-tance against cold stress, might play role as transcriptional

a

Fig. 7. qPCR expression analysis of BrBZR TFs after cold, salt, drought and ABA treatment (0e48 h) in B. rapa (aed). The error bars represent the standard error of the means of threeindependent replicates.

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activators to regulate CBF cold responsive pathway which ulti-mately increase cold/freezing tolerance in B. rapa (Fig. 9).

We identified some candidate genes that may play a role inresponses to drought and salt stress based on their expressionpatterns. Most of the BrBZR TFs were up-regulated in response toexogenous ABA treatment. Specifically BrBZR1-1, BrBZR1-2,BrBEH10, BrBES1-1, BrBES1-2, and BrBES1-3 exhibited up-regulation in response to ABA treatment. The proteins encodedby these genes interact with the ABA-responsive protein GRF10 (a14-3-3 PROTEIN G-BOX FACTOR14 EPSILON/GF14) in addition toparticipating in BR signaling (Schultz et al., 1998, Fig. 6). In ourprotein interaction study, these six BrBZR proteins also showedstrong associations with DWF4, which functions in the response tojasmonic acid in addition to BR signaling (Kim et al., 2013). Like-wise, BIN2, a member of plant glycogen synthase kinase (GSK)family, plays important roles in BR signaling (Jonak and Heribert,

2002). As shown in Fig. 8, BIN2 is also strongly associated withthe BrBZRs. AtSK1 (Arabidopsis thaliana SHAGGY KINASE 1), amember of the GSK family that is closely associated with BIN2, isinvolved in salt, drought, and exogenous ABA resistance (Jonak andHeribert, 2002; Piao et al., 1999). Our results suggest that most ofthe BrBZR TFs appear to have stress resistance activity in addition toparticipating in BR signaling processes. Additionally, their wide-spread expression in different tissues suggests that they couldfunction in plant growth and development.

5. Conclusion

It can be concluded that, this is the first comprehensive, sys-tematic analysis of the BZR TFs, including tissue-specific transcriptprofiling and stress responses of all members of this importantfamily. In addition to their BR responsiveness in B. rapa, these TFs

b

c

Fig. 7. (continued).

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d

Fig. 7. (continued).

Fig. 8. Interaction network of 15 BrBZR TFs identified in B. rapa and related genes in Arabidopsis. Stronger associations are represented by thicker lines.

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Fig. 9. The speculative cold tolerance pathway of Brassinazole Resistant TFs (BZRs) inB. rapa: where BrBZRs acting as transcriptional activators at Inducer of CBF Expression(ICE) level, function upstream of the CBF regulon.

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represent candidates to play roles in organ development, especiallyflower development, as indicated by their expression patternsduring flower bud development. From a hypothesis of having stressresistance function of BZR TFs, we systemically studied stressresistance related expression profiles of all BrBZRs, where 6 and 11BrBZRs against salt and drought stresses showed differential tran-script abundance levels. Specifically, all BrBZRs were induced byexogenous ABA treatment. More importantly, all BrBZRs showeddifferential expression against low temperatures and they wereproposed as transcriptional activators of CBF cold responsepathway of B. rapa. Notably, three BrBZRs (BrBEH4, BrBEH5 andBrBEH6) gave co-responsive expression against all four stresses,suggesting that these genes might have multiple stress resistancerelated functions in B. rapa. Thus, our findings in this study shouldfacilitate the selection of appropriate candidate genes for furtherfunctional characterization. Specially, the highly induced BrBZR TFsmight be potential resources for developing stress resistant varietyof Brassica crops applying molecular breeding techniques againstabiotic stresses.

Author contributions

The work presented here was carried out in collaborationamong all authors. Gopal Saha carried out the computationalanalysis, plant culture and sample preparation, performed RT-PCRand real-time PCR, analyzed the data and drafted the manuscript.Jong-In Park and Hee-Jeong Jung collected primary data regardinggenes and cultured plants and collected samples for organ study.Nasar Uddin Ahmed and Md. Abdul Kayum designed the stressexperiments and cultured the plants and gave stress treatments tothe two B. rapa inbred lines ‘Chiifu’ and ‘Kenshin’. Jong-Goo Kangrevised the final version of the manuscript and gave suggestions forimproving it. Ill-Sup Nou designed and participated in all the ex-periments and assisted in improving the technical sites of theproject. All authors have read and approved the final manuscript.

Acknowledgments

This research was supported by Golden Seed Project (Center forHorticultural Seed Development), Ministry of Agriculture, Food andRural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF),

Rural Development Administration (RDA) and Korea Forest Service(KFS) (Grant No. 213003-04-3-SB110).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2015.04.013.

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