Plant Physiology and Biochemistry 92 (2015) 92e104

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Research article

Molecular characterization of BZR transcription factor family and abiotic 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

a b s t r a c t

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

BRASSINAZOLE-RESISTANT (BZR) transcription factors (TFs) are primarily well known as positive regulators of Brassinosteroid (BR) signal transduction in different plants. BR is a plant specific steroid hormone, which has multiple stress resistance functions besides various growth regulatory roles. Being an important 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 and characterized 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 a high degree of similarity also observed with recognized BZRs of other plant species from the comparison studies. In the organ specific expression analyses, 6 BrBZR TFs constitutively expressed in flower developmental stages indicating their flower specific functions. Subsequently, from the stress resistance related expression profiles differential transcript abundance levels were observed by 6 and 11 BrBZRs against salt and drought stresses, respectively. All BrBZRs showed several folds up-regulation against exogenous ABA treatment. All BrBZRs also showed differential expression against low temperature stress treatments and these TFs were proposed as transcriptional activators of CBF cold response pathway of B. rapa. Notably, three BrBZRs gave co-responsive expression against all the stresses tested here, suggesting their multiple stress resistance related functions. Thus, the findings would be helpful in resolving the complex regulatory mechanism of BZRs in stress resistance and further functional genomics study of these potential TFs in different Brassica crops. © 2015 Published by Elsevier Masson SAS.

Keywords: Abiotic stress BEH Brassica rapa BZR TFs

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

Abbreviations: BZR, BRASSINAZOLE-RESISTANT; BEH, BRASSINAZOLE-RESISTANT Homolog; TFs, transcription factors; BR, brassinosteroid; Br, Brassica rapa; BRAD, Brassica database; CDS, coding DNA sequence; DUF, domain of unknown function; bHLH, basic helix-loop-helix; RT-PCR, reverse transcription-polymerase chain reaction; qPCR, quantitative polymerase chain reaction; PIR, protein information resource; mL, micro-litre. * Corresponding author. E-mail address: [email protected] (I.-S. Nou). http://dx.doi.org/10.1016/j.plaphy.2015.04.013 0981-9428/© 2015 Published by Elsevier Masson SAS.

physiological processes including cell elongation, cell division, senescence, vascular differentiation, reproduction, photomorphogenesis and, responses to various environmental cues (Clouse et al., 1996; Li and Chory, 1997; Ye et al., 2010; Clouse, 2011). Brassinosteroids (BRs), as stress alleviators dispersed ubiquitously in the plants, 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 transcriptional network through which BZR TFs control BR responses have been revealed by identification and characterization of BZR target genes in different studies (Yu et al., 2008, 2011; Luo et al., 2010; Sun et al., 2010). Now-a-days, different TF families have been characterized as stress responsive besides their growth regulatory functions. Being an important factor in the BR synthesis pathway, BZR

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TFs might have stress resistance activities. Surprisingly, no study regarding stress resistance of BZR TFs in any crop plant has been reported to date. Therefore, we conducted this study to identify and characterize stress regulatory functions of BZR TFs in B. rapa. BRASSINAZOLE-RESISTANT 1 (BZR1) and BRASSINOSTEROIDINSENSITIVE 1-EMS-SUPPRESSOR 1 (BES1) make up a small gene family with at least four other members in Arabidopsis, including BEH1e4. All six members of this plant-specific family likely have partially redundant functions in BR signaling (Yin et al., 2005; He et al., 2005). Like BES1 and BZR1, BEH1e4 exist in both phosphorylated and dephosphorylated form and accumulate in dephosphorylated form in response to BR treatment (Yin et al., 2005). Dissection of the functional domains of BZR proteins has revealed highly conserved N-terminal domains that have DNA binding activity both in vitro and in vivo (Yin et al., 2005; He et al., 2005). The BZR1 DNA binding domain (encoded by the first exon) is the most conserved region of the BZR1-related proteins and is thus named the BZR domain (He et al., 2005). BZR1 and BES1/BZR2 are two well-characterized transcription factors in the BR signaling pathway. These transcription factors are unique to plants and share high 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 basic helix-loop-helix (bHLH) DNA binding motif in the N terminus and bind to E-box (CANNTG) and BRRE (CGTGT/CG) elements, respectively (He et al., 2005; Yin et al., 2005). The central regions of these proteins contain 22e24 putative phosphorylation sites for the GSK3 kinase family, and several contain a putative PEST motif involved in protein degradation (Yin et al., 2005). The genus Brassica includes a number of important crops that serve as a source of oil, vegetables, condiments, dietary fiber, and vitamin C (Talalay and Fahey, 2001). Among the species in this genus, B. rapa comprises several important subspecies, such as Chinese cabbage (B. rapa ssp. pekinensis), non-heading Chinese cabbage (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’ genome and was therefore selected for genome sequencing (Brassica Genome Gateway: http://brassica.bbsrc.ac.uk; The Korea Brassica rapa Genome Project: http://www.brassica-rapa.org/BRGP/index. jsp). Comparative genomic analysis confirmed that B. rapa underwent genome triplication since its divergence from Arabidopsis (Song et al., 2013). This species has already proven to be a useful model for studying polyploidy because it has a relatively small genome (approximately 529 megabase-pairs [Mbp]) compared with other Brassica species. Numerous studies have focused on the regulatory roles of BZR TFs in BR signal transduction in Arabidopsis, a close relative of B. rapa. However, the stress responsiveness of BZR TFs in any crop species remains to be investigated. In this study, we selected B. rapa as an important vegetable crop worldwide that is subjected to a variety of abiotic stresses to investigate the stress responsiveness of BrBZR TFs. The recent sequencing of the B. rapa ssp. pekinensis genome (Wang et al., 2011) offers the possibility of genome-wide analysis of BZR TFs. In this study, we analyzed the genomic localizations, protein motif structures, phylogenetic relationships, and structures of candidate BZR TFs in B. rapa. In addition, we carried out genomewide expression profiling of these genes in plants subjected to lowtemperature treatment involving the cold-tolerant and -susceptible inbred B. rapa lines ‘Chiifu’ and ‘Kenshin’, respectively. We further investigated the expression patterns of these TFs in response to salt, drought and ABA treatment and examined their tissue-specific expression in different organs. We observed constitutive expression of several BrBZR TFs throughout the flower bud development stages. We identified BrBZR TFs with co-responsive expressions against stress treatments, suggesting that they play multiple roles

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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) was conducted using keyword ‘BZR’ (http://brassicadb.org/brad/; Cheng et al., 2011). Protein sequences and CDS of the identified B. rapa BZR TFs 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 used to confirm the identity of the BZR domain, and a protein homology study was conducted using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm the putative BZR TFs in B. rapa. The primary structures of the genes were analyzed using protParam (http://expasy.org/tools/protparam. html). Multiple protein sequences were aligned using PIR (http:// pir.georgetown.edu/pirwww/search/multialn.shtml).The number of introns and exons was determined by aligning the CDS with the genomic sequences using the Gene Structure Display Server (GSDS) web tool (Guo et al., 2007). The subcellular localization of BrBZR proteins was predicted using ProtComp 9.0 from Softberry (http:// linux1.softberry.com/berry.phtml). To identify the putative cisacting regulatory elements of BrBZR TFs, about 1500-bp CDS sequences from the translation initiation codon (ATG) was analyzed using 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 and Arabidopsis using ClustalX (Thompson et al., 1997). Phylogenetic trees were generated with MEGA6.06 using the Neighbor-Joining (NJ) algorithm (Tamura et al., 2013). Bootstrap analysis with 1000 replicates was used to evaluate the significance of the nodes. Pairwise gap deletion mode was used to ensure that the divergent domains could contribute to the topology of the NJ tree. 2.3. Analysis of conserved motifs in BZR TFs The BZR protein sequences were analyzed using MEME software (Multiple Em for Motif Elicitation, V4.9.0) (Bailey et al., 2006). A MEME search was executed with the following parameters: (1) optimum motif width 6 and 200; (2) maximum number of motifs 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 duplicate genes, where both the similarity and query coverage percentage of the candidate genes were >80% (Kong et al., 2013). Positional information about all candidate BZR TFs along the 10 chromosomes of B. rapa was obtained from the Brassica database (http://brassicadb. org/brad/; Cheng et al., 2011). The positions of the genes along 10 chromosomes and duplication lines among genes on the chromosomes were drawn manually. 2.5. Collection and preparation of plant material B. rapa ‘SUN-3061’ plants were grown at the Department of Horticulture, Sunchon National University, Korea. For the organ study, fresh roots, stems, leaves, and flower buds were harvested, frozen immediately in liquid nitrogen, and stored at 80  C for RNA

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isolation. For the three abiotic stress treatment, two inbred lines of B. rapa ssp. pekinensis, i.e., ‘Chiifu’ and ‘Kenshin’, were used. ‘Chiifu’ originated in temperate regions, whereas ‘Kenshin’ originated in subtropical and tropical regions (Lee et al., 2013). Plants were cultivated under aseptic conditions in semi-solid medium for four weeks. Three stress treatments, including cold, drought, and salt, were administered for various durations (0 h, 1 h, 4 h, 12 h, 24 h and 48 h). Plant samples were transferred to an incubator at 4  C to induce cold stress. Drought/desiccation stress was simulated by drying the plants on Whatmann 3 mm filter sheets. To induce salt and ABA stress, plant samples were transferred to rectangular Petri dishes (72  72  100 mm) containing 200 mM NaCl and 100 mM abscisic 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 analysis of 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 cDNA from the plant and flower organs as templates in master mixes composed of 20 pmol each primer, 150 mM each dNTP, 1.2 U Taq polymerase, 1 Taq polymerase buffer, and double-distilled H2O in a total volume of 20 mL in 0.5-mL PCR tubes. The samples were subjected to the following conditions: pre-denaturing at 94  C for 5 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 a final extension for 5 min at 72  C. 2.7. Quantitative PCR expression analysis Real-time quantitative PCR was performed using 1 mL cDNA in a 10-mL reaction volume employing iTaqTM SYBR® Green Super-mix with ROX (California, USA). The specific primers used for real-time PCR are listed in Supplementary Table 3. The conditions for realtime PCR were as follows: 5 min at 95  C, followed by 40 cycles at 95  C for 10 s, 55  C for 10 s, and 72  C for 15 s. The fluorescence was measured following the last step of each cycle, and three replications were used per sample. Amplification detection and data analysis were conducted using LightCycler96 (Roche, Germany).

3. Results 3.1. Sequence analysis and phylogenetic classification of BZR TFs in B. rapa We identified 15 BZR TFs in B. rapa, which we designated B. rapa BZR (BrBZR) TFs. The predicted sizes of the 15 BrBZRs ranged from 152 to 386 amino acids (16.46e42.53 kDa), and the predicted isoelectric points varied from 4.44 to 9.48 (Table 1). Analysis of the protein domain organization showed that 13 BrBZR TFs contained the ‘DUF822’ domain, which is characteristic of BZR TFs. The remaining two proteins exhibited other distinguishing BZR TFs features but lacked the ‘DUF822’ domain. Additionally, the DNA sequences of the 15 BrBZR TFs were determined based on the B. rapa whole-genome sequence. Analysis of the intron and exon distribution showed that most of the genes exhibited similar splicing patterns (Fig. 1). A BLAST search of the NCBI database to compare the 15 BrBZRs with BZRs of other species revealed that the deduced amino acid sequences of all 15 BrBZRs shared the highest similarity levels with Arabidopsis BZR TFs. The sequence similarity ranged from 50 to 94%, more specifically, 12 BrBZRs shared greater than 80% similarity with Arabidopsis BZR TFs (Table 2). The similarity among the BrBZR TFs sequences ranged from 57 to 96%, and 11 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 BZR TFs from NCBI and constructed a phylogenetic tree with 15 deduced amino acid sequences of BrBZR using the NJ method (Fig. 3). In the phylogenetic tree, we also identified close homologs of Arabidopsis BZR and BZR-homolog (BEH). Two BrBZR TFs formed a tight group with BZR1, which we subsequently designated BrBZR1-1 and BrBZR1-2. Accordingly, three TFs closely related to BZR2/BES1 were designated BrBES1-1, BrBES1-2, and BrBES1-3. The remaining 10 BZR TFs were closely grouped with Arabidopsis BZR1-homologs (BEH) and were therefore designated BrBEH1e10. All of the rice BZR TFs exhibited distant relationships with both B. rapa and Arabidopsis BZR TFs, especially with the BZR1 and BZR2/BES1 cluster in the phylogenetic tree. This result suggests that expansion of BZR1 and BZR2/BES1 took place after the divergence of dicots and monocots. Motif searching identified 10 motifs which are characteristic to BrBZR TFs (Fig. 1 and Supplementary Table 1). Motif searching and multiple sequence alignments revealed that most of the TFs have an atypical basic helix-loop-helix (bHLH) DNA-binding motif in their N

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

BrBZR1-1 BrBZR1-2 BrBES1-1 BrBES1-2 BrBEH1-3 BrBEH1 BrBEH2 BrBEH3 BrBEH4 BrBEH5 BrBEH6 BrBEH7 BrBEH8 BrBEH9 BrBEH10

Locus ID

Bra003772 Bra008378 Bra010093 Bra011706 Bra008177 Bra012570 Bra012891 Bra013359 Bra015868 Bra016508 Bra017782 Bra020986 Bra025733 Bra031077 Bra035060

Located chromosome number

ORF (bp)

A07 A02 A06 A01 A02 A03 A03 A01 A07 A08 A03 A08 A06 A09 A07

996 945 1002 1015 1161 879 924 675 831 831 459 912 942 857 636

Protein Length (aa)

BZR domain starteend (aa)

PI

MW (kDa)

331 314 333 331 386 292 307 224 276 276 152 303 313 285 211

17e170 16e160 16e171 17e164 81e223 2e153 2e155 None 2e141 2e141 None 9e152 14e169 15e166 7e111

9.05 9.40 9.26 9.44 9.41 8.72 7.57 4.99 8.52 8.69 4.44 8.63 9.16 9.48 5.43

35.87 34.34 36.17 36.18 42.53 31.41 33.06 24.04 30.03 30.05 16.46 33.09 33.73 30.74 22.41

Sub-cellular localization

No. of introns

N N N N N N N EC N N EC N N N N

1 1 1 1 2 1 1 2 1 1 0 1 1 1 1

Abbreviations: ORF, open reading frame; bp, base pair; aa, amino acid; kDa, kilo Dalton; PI, iso-electric point; MW, molecular weight; N, nuclear; EC, extracellular. a Brassica database (http://brassica.org/brad/index.php).

Fig. 1. Schematic representation of motifs and intro-exon distribution identified in B. rapa BZR proteins. Different motifs are indicated by different colors, and the names of all members are shown on the left side of the figure, along with their phylogenetic relatedness. On the right, the introneexon organization patterns of 15 BrBZR TFs are shown, along with their intron splicing patterns. (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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termini represented by motif 1 (Figs. 1 and 4). The middle portions of these TFs harbor serine (S)-rich phosphorylation sites and a PEST domain, which are represented by motif 6 and motif 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 highly conserved among all of the proteins. Although BrBEH3 and BrBEH6 lack motif 1, they possess four other characteristic motifs of the BZR family. Among the 15 BrBZR TFs, 12 contain a single intron with similar splicing patterns (‘0’), while the splicing pattern of BrBEH10 is ‘2’. Among the remaining three TFs (BrBES1-3, BrBEH3, and BrBEH6), BrBES1-3 and BrBEH3 have double introns, while BrBEH6 lacks any introns (Fig. 1). We predicted subcellular localization of the 15 BrBZR TFs using Protcomp 9.0 from Softberry. As transcription factor family proteins, all but two BrBZRs were identified to have nuclear localization (Table 1). The exception is in case of BrBEH3 and BrBEH6 and they were found to be located as extracellular proteins of B. rapa. To identify the putative cis-acting regulatory elements in BrBZR TFs, about 1500-bp CDS sequences from the start codons (ATG) were critically examined. The identified cis-elements included abiotic stress responsive elements, as well as those involved in light and circadian rhythm regulation, disease resistance and seed development, such as: TC-rich repeats, TCA element, HSE element, ERE element, Box-W1 element, MBS element, LTR element, CGTCA motif, TGACG-motif, TGA element, ABRE, CE3, motif IIb, GAREmotif, P-box, SARE, AuxRR-core, GC-motif, WUN-motif and circadian (Supplementary Table 2). 3.2. Organ-specific expression analysis under unstressed conditions We performed RT-PCR analysis using specific primers with equal amounts of cDNA templates prepared from the mRNA of roots, stems, leaves, and flower buds of healthy, unstressed Chinese cabbage plants to examine the organ-specific expression patterns of the BrBZR TFs. BrBZR1-1, BrBZR1-2, BrBES1-1, BrBES1-2, BrBEH1, and BrBEH4e7 were ubiquitously expressed in all organs examined, with variable transcript levels (Fig. 5a). Specifically, all TFs except BrBEH2 were expressed in the flower buds of B. rapa. BrBEH2 was not expressed in any of the tissues examined. We further investigated the expression of all BrBZR TFs in flower buds at six stages of development (from very young to mature buds). All TFs had distinct expression patterns at all developmental stages. Intriguingly, the expression of BrBES1-1, BrBEH1, BrBEH3, BrBEH7, and BrBEH8 gradually increased until the mature bud stage (Fig. 5b). By contrast, the expression of BrBEH2 gradually increased to developmental stage four (S4) and decreased gradually in stages five and six. These six TFs may play regulatory roles in flower bud development in B. rapa. 3.3. Expression analysis under abiotic stress conditions 3.3.1. Microarray analysis of gene expression in response to cold and freezing stress We analyzed microarray expressions of 15 BrBZRs from our previously published data set, where two contrasting inbred lines of B. rapa, Chiifu and Kenshin were treated with cold and freezing stresses (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 originated in subtropical and tropical regions. Therefore, they are expected to respond differently to cold and freezing temperatures. In nature, Kenshin exhibits severe damage upon exposure to low temperatures, while Chiifu does not exhibit such damage (Dong et al., 2014). Moreover, Kenshin has been used as a breeding stock to develop

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Table 2 Homology 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. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

BrBZR1-1 BrBZR1-2 BrBES1-1 BrBES1-2 BrBES1-3 BrBEH1 BrBEH2 BrBEH3 BrBEH4 BrBEH5 BrBEH6 BrBEH7 BrBEH8 BrBEH9 BrBEH10

NP_565099 NP_565099 NP_973863 NP_973863 NP_973863 NP_565187 NP_565187 NP_565187 NP_193624 NP_193624 NP_193624 Q94A43 Q94A43 NP_190644 NP_565099

Brassinazole-resistant 1 protein Brassinazole-resistant 1 protein Protein brassinazole-resistant 2 Protein brassinazole-resistant 2 Protein brassinazole-resistant 2 BES1/BZR1 homolog protein 4 BES1/BZR1 homolog protein 4 BES1/BZR1 homolog protein 4 BES1/BZR1 homolog protein 3 BES1/BZR1 homolog protein 3 BES1/BZR1 homolog protein 3 BES1/BZR1 homolog protein 2 BES1/BZR1 homolog protein 2 BES1/BZR1 homolog protein 1 brassinazole-resistant 1 protein

92% 81% 86% 88% 78% 86% 84% 63% 93% 93% 94% 91% 90% 87% 50%

0 5.00E-161 1.00E-167 9.00E-177 5.00E-163 2.00E-152 4.00E-155 1.00E-36 8.00E-164 4.00E-165 2.00E-75 2.00E-167 2.00E-166 6.00E-140 3.00E-21

A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.

Theologis et al., 2000 Theologis et al., 2000 Theologis et al., 2000 Theologis et al., 2000 Theologis et al., 2000 Theologis et al., 2000 Theologis et al., 2000 Theologis et al., 2000 Mayer et al., 1999 Mayer et al., 1999 Mayer et al., 1999 Bevan et al., 1998 Bevan et al., 1998 Salanoubat et al., 2000 Theologis et al., 2000

A01

A02

A04

A03

A05

A06

thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana

A08

A07

A09

A10

BrBEH7 (+)

BrBEH5 (+)

BrBES1-2 (+) BrBEH6 (+) BrBEH10 (+) BrBEH1 (+)

BrBEH3 (-) BrBZR1-1 (-) BrBEH9 (-) BrBEH4 (+)

BrBES1-1 (-)

BrBZR1-2 (+) BrBEH2 (+)

BrBEH8 (-) BrBES1-3 (-)

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 names represent 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 orientation of 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 (in Megabases; 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.)

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 expression patterns between the two lines, while the remaining genes exhibited little or no expression. BrBEH4 was differentially expressed between the two lines. BrBEH6, BrBEH7, and BrBEH8 exhibited variable expression patterns at different temperatures in “Chiifu”. Moreover, in “Chiifu”, BrBZR1-1 and BrBES1-3 were more highly expressed in response to cold (0 and 4  C) versus freezing temperatures. By contrast, in ‘Kenshin’, BrBEH8 was highly expressed upon exposure to both cold and freezing temperatures. In summary, the BrBEH TFs were more highly induced than the BrBZR1 and BrBES1 TFs in response to cold or freezing stress. 3.3.2. Quantitative PCR analysis of gene expression under abiotic stress conditions One of our main objectives was to identify BrBZR TFs that are stress responsive in addition to playing various roles in plant growth. 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 cold stress, we also examined the salt, drought, and ABA responsiveness of 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 in response to cold stress; the expression of these TFs gradually increased from 0 h to 12 h and immediately decreased at 24 h. Among these TFs, only BrBEH4 and BrBEH6 were up-regulated at 48 h BrBEH1, BrBEH2, BrBEH3, and BrBEH5 were up-regulated at 12 h and were expressed at (approximately) control levels at the other time points. BrBEH7 was down-regulated from the very beginning of stress treatment. Conversely, in ‘Kenshin’, most of these TFs did not show any significant changes in expression, or were 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 response to salt treatment until the middle stage of treatment, followed by down-regulation to minimum levels, which were maintained

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BrBZR1-2 and BrBES1-3 were down-regulated during the early stage of hormone treatment, which was followed by up-regulation until 48 h of treatment. The remaining BrBZR TFs were up- or down-regulated up to the last stage of ABA treatment, except for BrBEH2, which was down-regulated during the later hours of treatment 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 cold stress. The highly expressed BrBZR TFs in ‘Chiifu’ might be involved in cold resistance, while their inactivity or very low activity in ‘Kenshin’ might play a role in the cold susceptibility of this line. We also identified some candidate BrBZR TFs that showed considerable responsiveness against salt, drought, and ABA stress in B. rapa. We found that BrBEH4, BrBEH5 and BrBEH6 exhibited similar expression patterns in response to all four stresses, suggesting that these TFs might have multiple stress resistance-related functions in B. rapa. 3.4. Analysis of BrBZR protein interactions

Fig. 3. Phylogenetic tree showing the relatedness of the deduced full-length amino acid 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 acid substitutions between sequences as determined by the Poisson evolutionary distance method. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

We investigated the interactions of the 15 B. rapa BZR TFs and their association with Arabidopsis proteins, including their functional 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 Arabidopsis, respectively, are involved in a brassinosteroid (BR) signaling network. BRASSINOSTEROID-INSENSITIVE 2 (BIN2), BRASSINOSTEROID INSENSITIVE 1, BRI1 SUPPRESSOR 1 (BSU1), EARLY FLOWERING 6 (ELF6), RELATIVE OF EARLY FLOWERING 6 (REF6), GENERAL REGULATORY FACTOR 8 (GRF8), GENERAL REGULATORY FACTOR 10 (GRF10), DWARF 4 (DWF4), BES1-INTERACTING MYCLIKE 1 (BIM1), and HIGH NITROGEN INSENSITIVE 9 (HNI9) appear to participate in this process. The six above-mentioned B. rapa BZR TFs have evolved as functional partners in this interaction network. We found that these five B. rapa BZR TFs are responsive to ABA and are strongly associated with Arabidopsis GRF10 and DWF4, which are involved in ABA and jasmonic acid resistance, respectively (Ren et al., 2009; Kim et al., 2013). The 10 remaining B. rapa BZR homolog TFs (BrBEH1e9) are highly homologous to four unknown Arabidopsis proteins (AT4G18890, AT4G36780, AT1G78700, and AT3G50750), which might play positive regulatory roles in brassinosteroid signaling, like BZR1; Yin et al. (2005) reported their genetic redundancy (identified as BZR homologs) in BR regulation. The tissue-specific and stress-responsive expression patterns of these TFs were similar to those of BrBZR1 and BrBES1. 4. Discussion

through the end of treatment. By contrast, BrBES1-3 and BrBEH9 were down-regulated from the beginning of stress treatment, becoming inactive at 48 h. The expression levels of the remaining TFs nearly matched those of the control (Fig. 7b). We identified some BrBZR TFs as candidates that might be involved 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 both the early and late hours of stress treatment. Conversely, the downregulation of BrBES1-3 and BrBEH9 throughout the stress period is also notable. These TFs became inactive in the final hour of stress treatment; their susceptibility to drought stress might have some connection with the stress resistance of B. rapa ‘Chiifu’ (Fig. 7c). Most of the BrBZR TFs in B. rapa ‘Chiifu’ were up-regulated in response to ABA treatment. Specifically, BrBZR1-1 and BrBES1-2 were gradually up-regulated until 48 h of ABA treatment.

The BZR TF family appears to be involved in the regulation of various processes in plants. While major studies have revealed the positive 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 TF family has previously been reported. Complete and accurate annotation of genes/proteins is an essential starting point for functional studies of transcription factor families. In our current genome-wide study of B. rapa, we investigated the organ-specific expression of 15 BrBZR TFs; their ubiquitous expression in most organs suggests that they might function as growth regulators during B. rapa development. A more thorough investigation revealed that these genes were differentially expressed during flower bud development (Fig. 5b). The function of these TFs in plant organ growth and development has not previously been reported. The expression patterns of these TFs during flower bud development makes them good candidates for further investigations of

98 G. Saha et al. / Plant Physiology and Biochemistry 92 (2015) 92e104 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 lines above 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 amino acid 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 growth stages (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 indicates expression levels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

their growth-related functions. More importantly, in the study against cold, salt, drought, and ABA stresses, we found differential expression patterns of all the BrBZR TFs. Notably, all 15 BrBZR TFs contained predicted abiotic stress responsive cis-acting elements in their sequences. Outcomes of our study might be the foundation work to undertake further functional studies on these potential BZR TFs, which ultimately can provide a wider basis for exploitation of these candidate genes for genetic engineering of B. rapa. Most of the BrBZR TFs were more highly up-regulated (by several fold) in response to cold stress in the cold-resistant line (‘Chiifu’) than in the cold-susceptible line (‘Kenshin’), suggesting that these genes might play a role in the cold resistance of B. rapa ‘Chiifu’. To uncover the cold resistance mechanism of BrBZR TF family, we investigated their sequence organization and found conserved atypical bHLH domain in the N-terminal region which

also has been reported by He et al. (2005). Moreover, their ciselement analysis in all the gene sequences revealed E-box binding element (CANNTG) which is designated as ‘MBS’ (MYB binding sites) (Supplementary Table 2). We speculate our candidate BrBZR TFs also respond against cold/freezing binding with the core element (CANNTG) of C-REPEAT BINDING FACTOR (CBF) and regulate cold responsive genes [COLD REGULATING (COR) GENE] in the downstream to increase cold/freezing tolerance of B. rapa. Jiang et al. (2011) in their study presented evidence for a CBF coldresponse pathway in non-heading Chinese cabbage (Brassica campestris ssp. chinensis L Makino). They showed, BrICE1, a bHLH type transcription factor, which regulates CBF-cold responsive pathway. In this study, we also want to propose that our candidate BrBZR TFs having atypical bHLH domain organization those showed resistance against cold stress, might play role as transcriptional

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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 three independent replicates.

activators to regulate CBF cold responsive pathway which ultimately increase cold/freezing tolerance in B. rapa (Fig. 9). We identified some candidate genes that may play a role in responses to drought and salt stress based on their expression patterns. Most of the BrBZR TFs were up-regulated in response to exogenous ABA treatment. Specifically BrBZR1-1, BrBZR1-2, BrBEH10, BrBES1-1, BrBES1-2, and BrBES1-3 exhibited upregulation in response to ABA treatment. The proteins encoded by these genes interact with the ABA-responsive protein GRF10 (a 14-3-3 PROTEIN G-BOX FACTOR14 EPSILON/GF14) in addition to participating in BR signaling (Schultz et al., 1998, Fig. 6). In our protein interaction study, these six BrBZR proteins also showed strong associations with DWF4, which functions in the response to jasmonic acid in addition to BR signaling (Kim et al., 2013). Likewise, 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 with the BrBZRs. AtSK1 (Arabidopsis thaliana SHAGGY KINASE 1), a member of the GSK family that is closely associated with BIN2, is involved in salt, drought, and exogenous ABA resistance (Jonak and Heribert, 2002; Piao et al., 1999). Our results suggest that most of the BrBZR TFs appear to have stress resistance activity in addition to participating in BR signaling processes. Additionally, their widespread expression in different tissues suggests that they could function in plant growth and development. 5. Conclusion It can be concluded that, this is the first comprehensive, systematic analysis of the BZR TFs, including tissue-specific transcript profiling and stress responses of all members of this important family. In addition to their BR responsiveness in B. rapa, these TFs

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c

Fig. 7. (continued).

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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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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. References

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

represent candidates to play roles in organ development, especially flower development, as indicated by their expression patterns during flower bud development. From a hypothesis of having stress resistance function of BZR TFs, we systemically studied stress resistance related expression profiles of all BrBZRs, where 6 and 11 BrBZRs against salt and drought stresses showed differential transcript abundance levels. Specifically, all BrBZRs were induced by exogenous ABA treatment. More importantly, all BrBZRs showed differential expression against low temperatures and they were proposed as transcriptional activators of CBF cold response pathway of B. rapa. Notably, three BrBZRs (BrBEH4, BrBEH5 and BrBEH6) gave co-responsive expression against all four stresses, suggesting that these genes might have multiple stress resistance related functions in B. rapa. Thus, our findings in this study should facilitate the selection of appropriate candidate genes for further functional characterization. Specially, the highly induced BrBZR TFs might be potential resources for developing stress resistant variety of Brassica crops applying molecular breeding techniques against abiotic stresses. Author contributions The work presented here was carried out in collaboration among all authors. Gopal Saha carried out the computational analysis, plant culture and sample preparation, performed RT-PCR and real-time PCR, analyzed the data and drafted the manuscript. Jong-In Park and Hee-Jeong Jung collected primary data regarding genes and cultured plants and collected samples for organ study. Nasar Uddin Ahmed and Md. Abdul Kayum designed the stress experiments and cultured the plants and gave stress treatments to the two B. rapa inbred lines ‘Chiifu’ and ‘Kenshin’. Jong-Goo Kang revised the final version of the manuscript and gave suggestions for improving it. Ill-Sup Nou designed and participated in all the experiments and assisted in improving the technical sites of the project. All authors have read and approved the final manuscript. Acknowledgments This research was supported by Golden Seed Project (Center for Horticultural Seed Development), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF),

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