Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage

bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.


Introduction
Cooperation between transcription factors and the core transcription enzyme, RNA polymerase, initiates gene expression in eukaryote organisms. Transcription factors comprise approximately 3.5-7.0% of the genome [1]. The plant genome has a number of transcription factors families, such as MYB, AP2, bHLH, WRKY, NAC, and MADS [2]. The bZIP (Basic Leucine Zipper) family is one of the largest and most diverse transcription factor families in plants [3,4]. All bZIP family members share a highly conserved domain, which contains a basic region and a Leu zipper, known as the bZIP domain. The basic region contains a N-x7-R/K-x9 motif that directly binds to DNA and determines its nuclear localization. The leucine zipper forms an amphipathic surface, which plays an important role in bZIP transcription factors dimerization [3,4,5]. In addition, bZIP transcription factors have other conserved motifs besides the bZIP domain to modulate their transcriptional activity [3,5]. For example, phosphorylation of a conserved Ser or Thr in the R-X-X-S/T motif in many subfamily A bZIP factors activates target gene expression [6,7,8,9].

Protein Properties and Phylogenetic Analysis
To verify the bZIP domain in putative BrbZIPs [41], online tools such as to search for conserved domains within a protein or coding nucleotide sequence (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi) [48], SMART (Simple Modular Architecture Research Tool, http:// smart.embl-heidelberg.de/), Pfam (http://pfam.xfam.org/) and HMMER (Profile hidden Markov models for biological sequence analysis, http://www.ebi.ac.uk/Tools/hmmer/) were used to perform bZIP domain predictions. Proteins which showed the presence of bZIP domain with confidence (E-value <1.0) were selected for further analysis.
The molecular weight (kDa) and isoelectric point (pI) of BrbZIPs were calculated by DNAstar. The conserved motifs and protein architecture were predicted by the MEME (Multiple Em for Motif Elicitation) tool (http://meme-suite.org/tools/meme) with parameters set: optimum motif width 6 and 200, maximum number of motifs 25 as previously described [35]. All AtbZIPs and BrbZIPs were aligned with the MUSCLE tool and the maximum likelihood trees were generated using MEGA 5.0 as previously described [9,35,49,50].
The CDSs (coding sequences) of the BrbZIP genes were also downloaded from Brassica rapa database (BRAD, http://brassicadb.org/brad/index.php). The CDSs of the BrbZIP genes were used as the queries for local BLAST to search against the whole genome assembly of Brassica rapa (B.rapa_Chromosome_V1.5). The genomic sequences of BrbZIP genes were then retrieved. The online Gene Structure Display Server (GSDS2.0, http://gsds.cbi.pku.edu.cn/) [52] was used to decipher the architectures of BrbZIP genes.

qRT-PCR (Real-Time Quantitative RT-PCR) Analysis
Total RNA was extracted from samples after treatments with TRIzol reagent (TaKaRa). Total RNA was then treated with RNase-free DNase (TaKaRa) to remove DNA, and used for reverse transcription with PrimeScript™ RT Master Mix (Perfect Real Time, TaKaRa). Then, real-time qRT-PCR was performed using a CFX96 real-time PCR machine (Bio-Rad, Hercules, CA, USA) and SYBR Premix Ex Taq kit (TaKaRa) to monitor double-stranded DNA products as previously described [8,9,49,50]. Data from real-time PCR was analyzed by the software (Bio-Rad CFX Manager) and the standard curve method (delta-delta ct value) was used for calculating the relative expression of experimental genes normalized to the expression of cabbage ACTIN2 (BrACTIN2/Bra037560) according to the manufacturer's instructions. The primers used for qRT-PCR are listed online in S1 Table.

Plasmid Construction
To construct Myc-tagged BrABI5a and Myc-tagged BrABI5b, full-length CDSs were amplified via RT-PCR at first. The sequence-confirmed, full-length CDSs of BrABI5a and BrABI5b were then cloned into BamHⅠ and SalⅠ sites of the binary vector p1307-6Myc as previously described [8,9,49,50].
To make GFP-tagged BrABI5a and BrABI5b (BrABI5a-GFP and BrABI5b-GFP), the CDSs were removed from p1307-6Myc-BrABI5a and p1307-6Myc-BrABI5b and then inserted into the Cam-35S-GFP vector between the BamHⅠ and SalⅠsites, resulting in a C-terminal fusion to GFP.
To express BrABI5a and BrABI5b in yeast, the CDSs of BrABI5a and BrABI5b were cloned into the pPC86 vector between SalⅠ and EcoRⅠ sites as previously described [8,9]. To delete the bZIP motif on BrABI5a and BrABI5b, we truncated BrABI5a (1-352 aa, BrABI5aΔbZIP) and BrABI5b (1-310 aa, BrABI5bΔbZIP) via PCR amplification. The products BrABI5aΔbZIP and BrABI5bΔbZIP were also inserted into the pPC86 vector between SalⅠ and EcoRⅠ sites respectively. The primers used to construct the plasmids are listed online in S2 Table. All plasmids were confirmed by sequencing to avoid cloning errors.

Subcellular Localization
The Cam-35S-BrABI5a-GFP and Cam-35S-BrABI5b-GFP vectors were introduced into the Agrobacterium tumefaciens strain GV3101 and then infiltrated into 5-to 6-week-old Nicotiana benthamiana leaves for transient expression as previously described [9,53]. The Agrobacterium strains were infiltrated at an OD 600 of 0.5. For microscopic analyses, leaf discs were cut 3 days after infiltration. Cells from the lower epidermis were analyzed at room temperature with 20% glycine as the imaging medium. GFP-fluorescence signals were examined under an inverted Zeiss LSM 510 META fluorescence confocal microscope.

Yeast One-Hybrid Assay
The yeast strain yWAM2 was used to perform a yeast one-hybrid assay. Yeast transformation and growth assays were performed according to the Yeast Protocols Handbook provided by Clontech. Briefly, pPC86-BrABI5a or pPC86-BrABI5b or pPC86-BrABI5aΔbZIP or pPC86-BrABI5bΔbZIP combined with either pRS315-6×ABRE-HIS or pRS315-HIS were transformed into the yeast strain yWAM2 with the lithium acetate/single-stranded carrier DNA/polyethylene glycol method. The transformed yeast cells were selected on synthetic complete medium lacking leucine and tryptophan (SC-LW). DNA binding and transactivation were determined by measuring the growth of serial dilutions of transformed yeast cells on synthetic complete medium lacking leucine, tryptophan and histidine (SC-LWH) for 2-3 days. All of these were performed as previously described [8,9].

Construction of BrABI5a and BrABI5b Transgenic Plants
The p1307-6Myc-BrABI5a and p1307-6Myc-BrABI5b constructs were transformed into the Agrobacterium tumefaciens strain GV3101 and then infiltrated into abi5-1 plants with the floral dip method. Seeds (T 0 ) from infiltrated plants were selected on MS medium containing 25 μg/L hygromycin (Roche). Homozygous T 3 plants (derived from different T 1 transformants) of abi5-1 harboring each construct were used for ABA inhibition of seed germination as previously described [8,9].
ABA inhibition of seed germination was performed as previously described [8,9,13,30,54]. Briefly, seeds of Wassilewskija (Ws-2), abi5-1 and transgenic plants harbouring 6Myc-BrABI5a or 6Myc-BrABI5b (abi5-1:: 6Myc-BrABI5a or abi5-1:: 6Myc-BrABI5b) were sterilized in a solution containing 20% sodium hypochlorite and 0.1% Triton X-100 for 10 min, washed five times with sterile water, and sown on MS medium (Phytotech) with 0.3% Phytagel (Sigma-Aldrich) with different concentrations of ABA (Sigma-Aldrich). The plates were incubated in growth chambers at 4°C for 4 days followed by incubation at 23°C under continual illumination. To quantify the percentage of seedlings with green cotyledons, seeds were sown on MS medium containing different concentrations of ABA and analyzed on the indicated days after stratification. For radicle emergence assays, seeds were sown on MS medium without sucrose and determined 3 days after stratification.

Phylogenetic Analysis of the bZIP Transcription Factor Family in Chinese Cabbage
To gain evolutionary insight into the phylogenetic relationship in bZIP transcription factors between Brassica rapa and Arabidopsis thaliana, we constructed a phylogenetic tree with 136 BrbZIP and 75 AtbZIP transcription factors (Fig 1) and classified them using a previously reported method [1,3,4,5,35,36,37,38]. Phylogenetic analysis indicated that 23 BrbZIPs were categorized into subfamily A (Fig 1 and S3 Table), 4 proteins in BrbZIPs were closely related to subfamily B, 9 closely related to subfamily C, 17 in subfamily D, 4 in subfamily E, 6 in subfamily F, 9 in subfamily G, 5 in subfamily H, 23 in subfamily I, and 35 in subfamily S (Fig 1 and S3 Table).

Chromosomal Location of BrbZIP Genes in Chinese Cabbage
To determine the chromosomal distribution of BrbZIP genes, we used MapChart 2.2. The identified 136 BrbZIP genes, except Bra040260/BrbZIP17, were mapped on the A01 to A10 BrbZIP135 and Bra026896/BrbZIP134, was categorized as putative subfamily I BrbZIPs members (S3 Table) and showed 100% identity with each other at the cDNA and genomic DNA sequence level. The other pair, Bra007274/BrbZIP8 and Bra007276/BrbZIP9, comprised two putative subfamily A BrbZIPs (S3 Table) that displayed 98.23% (match/nonmatch = 834/15) and 98.79% (match/nonmatch = 1222/15) identity with each other at the cDNA and genomic DNA sequence level, respectively.

Gene Structure Analysis of Subfamily A BrbZIP Genes
The overall exon/intron profile is an index that determines phylogenetic relationships within a particular gene family from different organisms [38,55]. We also investigated the intron and exon organization of subfamily A BrbZIP genes (Fig 2 and S2 Fig). As shown in Fig 2, 17 of subfamily A BrbZIPs contained introns. All 17 subfamily A BrbZIPs had 1-3 intron/introns within the basic region of the bZIP domain (Fig 2 and S2 Fig). Surprisingly, 10 of these 17 subfamily A BrbZIPs members showed a similar gene structure pattern to their Arabidopsis orthologs (Fig 2 and S3 Fig).

Protein Architecture of Subfamily A BrbZIP Factors
To investigate the evolutionary relationships between Arabidopsis thaliana and Brassica rapa, we found a total of 25 motifs (with E-value cutoff <e-1.0) [35], including the conserved bZIP domain (motif 1), in BrbZIPs (S4 Table, S3 Fig). The distribution of motifs in each member of subfamily A bZIPs (13 AtbZIPs and 23 BrbZIPs) was also depicted individually (Fig 3). Motif 3 was shared by most members of this subfamily (31 AtbZIPs and BrbZIPs). Moreover, motif 6 was shared by 27, motif 7 by 29, motif 9 by 24, motif 12 by 25, and motif 14 by 21 members of this subfamily (31 AtbZIPs and BrbZIPs). These conserved motifs were shared by more than 50% members of this subfamily (S5 Table). Interestingly, some of these conserved motifs (6, 7, 9, 12 and 14) were also specifically found in subfamily A (S5 Table). High similarity at the protein architecture level was also observed among orthologs of subfamily A bZIPs between Arabidopsis thaliana and Brassica rapa (Fig 3). All the motifs found in subfamily A AtbZIPs also appeared in subfamily A BrbZIPs. Some additional motifs (motif 18 and 20) were specifically present in subfamily A BrbZIPs (Fig 3 and S5 Table). The presence of these conserved motifs between subfamily A AtbZIPs and BrbZIPs indicate they may exert similar biological significance in plant signaling pathways.

Expression Profiles of Subfamily A bZIP Genes in Response to ABA in Chinese Cabbage
To examine the biological significance of subfamily A BrbZIPs in ABA signaling, we determined their expression profile in response to ABA stimulation. Induced expression (more than 2 fold) occurred in many subfamily A BrbZIPs starting 4 hr and persisted up to 24 hr following ABA treatment (Fig 4). Interestingly, some members were greatly induced (more than 50 fold Fig 4A) or mildly induced (approximately 30 fold, Fig 4B and 4C).A few of members were not stimulated by ABA with no more than a 2 fold increase at any testing time (Fig 4D). These ABA-induced BrbZIP genes are candidates to mediate ABA signaling.

Sequence Analysis of BrABI5a and BrABI5b in Chinese Cabbage
Expression of the two putative orthologs of ABI5 in Chinese cabbage, Bra005287/BrbZIP14 and Bra017251/BrbZIP13 (designated as BrABI5a and BrABI5b, respectively), was strongly induced after ABA treatment (Fig 4). BrABI5a and BrABI5b encode predicted proteins with 438 and 396 amino acids. The calculated molecular masses of BrABI5a and BrABI5b were 46.2 and 42.3 kDa, and the predicted pI values were 9.22 and 9.48, respectively. Motif analysis showed that BrABI5a and BrABI5b contain nearly all conserved regions of ABI5 and ABI5-like proteins [8,9], such as the four conserved phosphorylation sites including sequences (C1, C2, C3 and C4), a bipartite nuclear localisation signal, and the bZIP domain (Fig 4). Gene architecture of BrABI5a and BrABI5b also displayed high similarity to ABI5 [8,13], which has a large exon at 5' terminal region followed by three small exons interrupted by three small introns at 3' terminal region (Fig 2 and S2 Fig). BrABI5a, BrABI5b, BolABI5 and ABI5 were highly  Functional Analysis of Two ABI5-Like bZIP Transcription Factors in Brassica rapa continually increased until 20hr (Fig 5C and 5D). Moreover, BrABI5b expression was induced by drought and salt stress (Fig 5D). However, BrABI5a expression was not significantly stimulated by drought (no more than 2 fold) or salt stress (no more than 2 fold) treatments during the testing period (Fig 5C).

Subcellular Localization of the BrABI5a and BrABI5b
As shown in Fig 6G, there is a putative NLS observed at the C-terminus of BrABI5a (amino acids 340 to 373) and BrABI5b (amino acids 298 to 331), respectively. We determined the subcellular localization of BrABI5a and BrABI5b. Both GFP fused BrABI5a and BrABI5b localized exclusively to the nucleus (Fig 6A-6F). These data indicate that BrABI5a and BrABI5b are nuclear-localized proteins.

Transactivation and DNA-Binding Activity of BrABI5a and BrABI5b
We investigated whether BrABI5a and BrABI5b could directly activate ABRE-controlled gene expression. As displayed in Fig 7, BrABI5a and BrABI5b significantly induced HIS expression in yeast cells or LUC expression more than 3 fold in Arabidopsis leaf mesophyll protoplasts. Consistent with our previous observations on BolABI5 [9], deletion of the bZIP domain abolished BrABI5a and BrABI5b DNA binding and transactivation activity (Fig 7A and 7B). These results indicate that BrABI5a and BrABI5b possess DNA binding and transactivation activity.

Discussion
Phylogenic analysis reveals that Brassica rapa displays a close evolutionary or biological relationship to the model organism Arabidopsis thaliana [56,57]. Comparative genomic studies revealed that more than 60% of the genome assemblies between Arabidopsis thaliana and Brassica rapa are highly conserved [39,40,56,57,58]. Around 93% of the total predicted Brassica rapa gene families also appear in Arabidopsis thaliana [57]. A previous study found that qRT-PCR analysis of the expression pattern of subfamily A BrbZIP genes in response to ABA treatment. Eleven-day-old seedlings were treated with 0.1 mM ABA followed by sampling at 0, 4, 8, 12, 16, 20 and 24 h. The relative expression of the subfamily A BrbZIP genes was normalized to the expression of the cabbage ACTIN2 gene (BrACTIN2) and expressed relative to the level in mock-treated seedlings. The expression of Bra003254 after ABA treatment was also determined, which is adjacent to Bra003253/BrbZIP10 (only 1630 bp far from it) and encodes a putative DHHC zinc binding domain like proteins. doi:10.1371/journal.pone.0158966.g004 Functional Analysis of Two ABI5-Like bZIP Transcription Factors in Brassica rapa transcription factors families with a predictable ortholog in Arabidopsis thaliana are significantly over retained in Brassica rapa [57]. Moreover, genes associated with regulatory networks for environmental stimuli, such as salt, cold, or light, or hormonal responses, such as auxin, brassinosteroid or ABA, in Arabidopsis thaliana are also highly retained in Brassica rapa [39,40,57]. However, the method used by Hwanng et al., (2014) to classify BrbZIPs increase the Functional Analysis of Two ABI5-Like bZIP Transcription Factors in Brassica rapa difficulty to determine the biological significance of specific subfamily BrbZIPs members [1,3,4,5,35,36,37,38,41]. For example, the putative orthologs of Arabidopsis subfamily A members, ABI5, AREB1/ABF2, AREB2/ABF4 and AREB3 [3,17,18] are divided into group 6A, group 11, group 1A and group 12, respectively [41]. Here, we built a phylogenetic tree with 75 AtbZIPs and 136 BrbZIPs that show all bZIP subfamilies are highly conserved between Arabidopsis thaliana and Brassica rapa (Fig 1). Moreover, subfamily A members also display a high similarity in their gene and protein architecture (Fig 2 and Fig 3). Functional characterization of two representative genes from subfamily A members further support that the BrbZIPs categorization method employed in our study has increased reliability (Figs 5, 6, 7 and 8). Our findings indicate that a colinear relationship established between AtbZIPs and BrbZIPs provides an advantageous reference to predict and determine the biological function of BrbZIPs in future studies.
In the current study, 136 members of bZIP genes are encoded by the Brassica rapa genome and are distributed across all 10 chromosomes (S1 Fig). As the A01 to A10 and C01 to C09 represent chromosomes of Brassica rarpa and Brassica oleracea respectively [41], these genes may be localized inaccurately by Hwanng et al. (2014). Two bZIPs transcription factors that display high similarity to ABI5 was also observed in Brassica oleracea (data not shown). In terms of the U's Triangle, Brassica rapa (A genome) and Brassica oleracea (C genome) formed the Yeast lines yWAM2 expressing the indicated plasmids were grown on synthetic complete medium without Leu and Trp (SC-LW; left) and on synthetic complete medium without Leu, Trp, and His (SC-HLW; right). Yeast cells were incubated until the optical density at 600 nm reached 0.5 and then diluted 2-fold (×2), 10-fold (×10), 50-fold (×50), or 250-fold (×250) and used for assays. (B) Transactivation activity BrABI5a or BrABI5b in Arabidopsis leaf mesophyll protoplasts. Transactivation experiments were performed using protoplasts prepared from Col-0 leaves. Transfected cells were cultured for 16 h without or with 5μM ABA, and relative LUC activity was assayed according to the Dual-Luciferase Reporter Assay Protocol provided by Promega. The empty vector control was also included as a negative control. The values shown are average fLUC (firefly, Photinus pyralis, LUC) activities normalized to rLUC (sea pansy, Renilla reniformis, LUC) activities. BrABI5aΔbZIP and BrABI5bΔbZIP are forms of BrABI5a and BrABI5b that carries a deletion of the intact C-terminal bZIP region respectively.  Here, the colinear relationship between AtbZIPs and BrbZIPs will facilitate future study into the biological functions of different BrbZIPs subfamily members in Brassica rapa and its close relatives, such as Brassica oleracea and Brassica napus. In addition, future studies should investigate gene copy number variations of homologous genes and determine their biological significance and/or differences among Arabidopsis, Brassica rapa, Brassica oleracea, and Brassica napus.