BioMed Research International

BioMed Research International / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 4376598 | https://doi.org/10.1155/2016/4376598

Indeok Hwang, Ranjith Kumar Manoharan, Jong-Goo Kang, Mi-Young Chung, Young-Wook Kim, Ill-Sup Nou, "Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress", BioMed Research International, vol. 2016, Article ID 4376598, 18 pages, 2016. https://doi.org/10.1155/2016/4376598

Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress

Academic Editor: Yeon-Su Lee
Received14 Dec 2015
Revised24 Mar 2016
Accepted27 Mar 2016
Published23 May 2016

Abstract

Cabbages (Brassica oleracea L.) are an important vegetable crop around world, and cold temperature is among the most significant abiotic stresses causing agricultural losses, especially in cabbage crops. Plant bZIP transcription factors play diverse roles in biotic/abiotic stress responses. In this study, 119 putative BolbZIP transcription factors were identified using amino acid sequences from several bZIP domain consensus sequences. The BolbZIP members were classified into 63 categories based on amino acid sequence similarity and were also compared with BrbZIP and AtbZIP transcription factors. Based on this BolbZIP identification and classification, cold stress-responsive BolbZIP genes were screened in inbred lines, BN106 and BN107, using RNA sequencing data and qRT-PCR. The expression level of the 3 genes, Bol008071, Bol033132, and Bol042729, was significantly increased in BN107 under cold conditions and was unchanged in BN106. The upregulation of these genes in BN107, a cold-susceptible inbred line, suggests that they might be significant components in the cold response. Among three identified genes, Bol033132 has 97% sequence similarity to Bra020735, which was identified in a screen for cold-related genes in B. rapa and a protein containing N-rich regions in LCRs. The results obtained in this study provide valuable information for understanding the potential function of BolbZIP transcription factors in cold stress responses.

1. Introduction

Cabbage (Brassica oleracea L.) plants represent one of the major vegetable crops grown worldwide. Most crops of B. oleracea and its sister species Brassica rapa produce a range of phytochemicals with diverse functions for plant defense such as polyphenolic compounds, carotenoids, and glucosinolates [1, 2]. The draft genome sequences of B. oleracea (with the CC genome) and B. rapa (with the AA genome) were recently published [3, 4]. A total of 66.5% (34,237) of B. oleracea genes and 74.9% (34,324) of B. rapa genes were clustered. In total, 5,735 B. rapa-specific genes and 9,832 B. oleracea-specific genes among 45,758 protein coding genes were identified. The availability of published genome sequence for these crop plants facilitates studies of structural and functional genomics in agronomically important species.

Plant bZIP transcription factors play diverse roles in developmental and physiological processes and biotic/abiotic stress responses such as ABA signaling for osmotic stress responses during vegetative growth [5], seed germination and flowering time [6], glucose-ABA signaling [7], sugar signaling during metabolism [8], lipid stress responses [9], response to zinc deficiency [10], salicylic acid- (SA-) dependent plant systemic defense responses and the activation of jasmonic acid- (JA-) and ethylene (ET-) dependent defense mechanisms [11], anthocyanin accumulation during photo morphogenesis [12], floral patterning [13], auxin-mediated histone acetylation related AtbZIP11 [14], and ABA signaling related to stress tolerance [15]. As the focus of recent studies due to their importance as regulator of responses to the biotic and abiotic stresses, bZIP transcription factors have been identified in diverse plants. Based on the presence of the UARR and LCRs, 136 bZIPs were identified in B. rapa; 64 were found in cucumber based on predicted structural features, 92 in sorghum through genome-wide identification and characterization, 89 in rice according to their DNA binding specificity and amino acid sequences in basic and hinge regions, 131 in soybean based on the basic region of the bZIP domain and the presence of additional conserved motifs, 75 in Arabidopsis according to sequence similarities of their basic region and additional conserved motifs, and 141 in Hordeum vulgare [1622]. However, little is known about the genome-wide survey and expression patterns of bZIP transcription factors in B. oleracea. Among the BolbZIPs, the function of only one gene related with drought stress and ABA has been reported. Expression of BolABI5 was dramatically induced by drought stress and exogenous ABA [23]. Heterogeneous expression of BolABI5 rescued the ABA-insensitive phenotype of the Arabidopsis abi5-1 mutant during seed germination, suggesting that BolABI5 likely functions in positive regulation of plant ABA responses.

The bZIP domain includes a basic region and a leucine zipper located on a contiguous α-helix. An N-x7-R/K motif comprising ~16 amino acids constitutes the basic region, which binds DNA containing a nuclear localization signal. The leucine zipper is composed of leucine residue repeat and is positioned precisely at nine amino acids towards the C-terminus from the arginine in the basic region, creating an amphipathic helix. To bind DNA, two subunits adhere via interactions between the hydrophobic sides of their helices, which create a superimposed coiled-coil structure for homo- or/and heterodimerization. Plant bZIPs preferentially bind to specific sequences, namely, the A-box (TACGTA), C-box (GACGTC), and G-box (CACGTG), but there are also examples of nonpalindromic binding sites [21].

In this study, we identified 119 BolbZIP proteins using the consensus sequence of several bZIP proteins and classified them based on specific amino acid sequence, unique amino acid repeat regions (UARRs), and low complexity regions (LCRs). Additionally, transcriptome analysis related to cold stress responses using RNA sequencing provided valuable information for research into stress tolerance and molecular breeding in B. oleracea.

2. Materials and Methods

2.1. Database Searches for bZIP Transcription Factors in B. oleracea

The AtbZIP, BrbZIP, and BolbZIP amino acid sequences obtained from TAIR (http://www.arabidopsis.org/), BRAD (http://brassicadb.org/brad/), and Bolbase (http://ocri-genomics.org/bolbase/). To confirm the presence of bZIP domain, UARR and LCRs in putative AtbZIP, and BrbZIP and BolbZIP proteins, the Motif scan tool (http://myhits.isb-sib.ch/cgi-bin/motif_scan), SMART tool (http://smart.embl-heidelberg.de/), and Batch CD-search tool (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) were used. bZIP proteins that showed the presence of a bZIP domain, UARR, and LCRs with confidence (-value < 0.1) in the Motif scan tool and Batch CD-search tool were used for further analyses. Next, LCRs were identified using the SMART tool.

2.2. Plant Material and Cold Treatment

Seeds of B. oleracea (inbred lines “BN106” and “BN107”) were germinated in soil and then grown for approximately 3 weeks in a growth chamber at 25°C under long day condition (16 h day/8 h night). For cold treatment, the 5-week-old plants were transferred to a 4°C growth chamber under continuous light conditions. The plants were then treated with cold temperature at 4°C for 6 h, followed by 0°C for 2 h. Further, the plants were subjected to freezing treatment at −2°C for 2 h followed by 4°C for 6 h.

2.3. RNA Extraction and cDNA Synthesis

Total RNA was isolated from plant tissues using an RNA extraction kit (Qiagen, USA) according to the manufacturer’s protocol. Total RNA was treated with RNase-free DNase (Promega, USA) to remove the genomic DNA contamination. The quality of total RNA was checked using a nanoDrop Spectrometer (nD-1000 Spectrophotometer, Peqlab) and agarose gel electrophoresis. cDNA was then synthesized using Superscript II reverse-transcriptase (Invitrogen), after which 5 μL (about 2 μg) total RNA and 1 μL of oligo dT (500 μg/mL) were mixed in the reaction tube and then heated at 65°C for 10 min. The enzyme was then added into the tube and incubated at 42°C for 50 min. Finally, the reaction tube was incubated at 70°C for 15 min to inactivate the enzyme.

2.4. RNA Sequencing

Two cabbage lines, BN106 and BN107 which exhibit different sensitivity to cold stress, were used for RNA sequencing. Total RNA was extracted from leaves of BN106 and BN107 at 2 h in 0°C. The total RNA was isolated using TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. Total RNA (20 μg) from each sample, BN106_22°C and BN107_22°C (control) and BN106_0°C and BN107_0°C (treated), were used for Illumina sequencing (33 G 101 bp paired-end reads; Seeders, Republic of Korea). Transcripts of unigenes assembled from the total reads were validated by direct comparison with gene sequences in the Phytozome 15 (https://phytozome.jgi.doe.gov/pz/portal.html) using BLASTx (threshold -value ≤ ). The number of mapped clean reads for each unigene was counted and normalized using the DESeq package in R on two independent biological replicates. From the differentially expressed gene dataset, the transcripts of bZIP transcription factors were analyzed for up- and downregulated differentially expressed genes. BolbZIP sequence and RNAseq database sequences were aligned to each other using ClustalW with default parameters (http://www.genome.jp/tools/clustalw/).

2.5. RT-PCR and qRT-PCR

Quantitative real-time PCR (qRT-PCR) and reverse transcription PCR (RT-PCR) were conducted using cDNA from cold treated plants using primers specific for the BolbZIP gene (see Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/4376598). RT-PCR was conducted using cDNA of plants exposed to cold and freezing temperatures (22°C, 4°C, 0°C, and −2°C). The PCR procedure involved predenaturation at 95°C for 5 min followed by cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 min, and then a final extension for 5 min at 72°C. qRT-PCR was conducted by subjecting the samples to initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 30 s, and final extension at 72°C for 2 min. An actin primer set for B. oleracea was used for normalization of RT-PCR and qRT-PCR.

3. Results

3.1. Identification of bZIP Transcription Factors in B. oleracea

To search for bZIP transcription factors in B. oleracea, we used the conserved bZIP domain consensus sequences (Table S2) of several proteins as BLASTP queries against the Brassica database (http://brassicadb.org/brad/). In addition, homology searches using 136 BrbZIP proteins were performed [16]. A total of 126 BolbZIP candidates were initially obtained with a probability -value threshold of 0.05. To confirm the presence of a bZIP domain in the selected bZIP proteins, domain searches were performed with several tools (see Section 2). After exclusion of the proteins lacking a bZIP domain, 119 putative BolbZIP transcription factors were identified. The position of each candidate BolbZIP gene in B. oleracea chromosome data available at Bolbase (Version 1.0) was then determined.

Among 119 candidate BolbZIP genes, 112 were mapped on chromosomes C01–C09 (Figure 1). 14 genes of BolbZIP were mapped on C01, 12 genes on C02, 15 genes on C03, 23 genes on C04, 8 genes on C05, 7 genes on C06, 10 genes on C07, 12 genes on C08, and 11 genes on C09. In particular, 20% of the BolbZIP genes mapped to chromosome 4 (Table S3). In addition, 7 genes were found in scaffolds that have yet been mapped to chromosomes. Bol024237 was anchored on Scaffold000093, Bol019052 on Scaffold000133, Bol016607 on Scaffold000153, Bol004200 on Scaffold000329, Bol003614 on Scaffold000345, Bol001886 on Scaffold000417, and Bol000879 on Scaffold000492.

3.2. Classification of BolbZIP Transcription Factors

We have classified the BolbZIP transcription factors based on amino acid sequence similarity to 136 BrbZIP and 75 AtbZIP proteins previously reported (Table 1) [16]. For the majority of bZIP proteins, we found orthologous groups including counterparts from each species, although occasionally no BrbZIP or AtbZIP homologs were found. AtbZIP and BrbZIP homologs of the BolbZIP proteins are summarized in Table 1. The proteins were divided into 63 categories based on the amino acid sequence similarity (Table 1). Most categories included two or three BolbZIP and BrbZIP proteins but a single AtbZIP. Analysis of the amino acid sequences revealed that the similarity between BolbZIP, BrbZIP, and AtbZIPs ranged from 50% to 90%. Several BolbZIP proteins showed over 90% similarity to the corresponding AtbZIP. For example, the similarity among Bol010308, At3g12250, and At5g06950 was 91–94%. For other genes, the closest homologs (with over 90% amino acid homology) were between the BolbZIP and the BrbZIP such as Bol004832 and Bra004689. BolbZIP proteins were also classified according to the method by Hwang et al. [16] based on UARRs and LCRs, which were further divided into 9 groups: glutamine (Q), aspartic acid (D), proline (P), asparagine (N), serine (S), glycine (G) rich domain, transmembrane (Tm) domain, LCRs only, and no LCRs except bZIP domain (Table 2, Tables S4 and S5). BolbZIP proteins and their orthologs from B. rapa and A. thaliana were found in the same groups. For example, BolbZIP of category 1 and its homologs Bra004550 and At2g46270 were classified into group 3A. LCRs of group 11 (only LCRs present) bZIP proteins composed single and mixed repeat natural amino acids. Group 12 contained bZIP proteins with no LCRs or specific amino acid-rich regions.


IndexB. oleraceaIdentity 1 (%)Identity 2 (%)B. rapa homologsA. thaliana homologs
Bol numberLength (aa)GroupBra numberLength (aa)GroupAt numberLength (aa)Group

1Bol0008793113A9575Bra0045503793AAt2g462703823A
Bol0177423283A8070
Bol029580300117679

2Bol0048323001165, 98, 62, 6275, 64Bra00025636211At2g423803214A
Bol0018863061182, 75, 61, 6271, 65Bra0046893064BAt3g581203294A
Bra0073803184A
Bra00332030411

3Bol0051153431A62, 83,9283Bra0001953341AAt2g406203671A
Bol0068823561A59, 98, 8679Bra0045823561A
Bol0206043361A88, 66, 6766Bra0169803421A

4Bol005139617107953Bra01695962410At2g4095072110
Bol006897639106561

5Bol0042002811259, 83, 84, 83, 6064, 88Bra00459728112At2g4107026212
Bol0051462721274, 59, 60, 60, 9369, 60Bra00727428212At3g568502976B
Bol0069022391293, 59, 60, 61, 7064, 58Bra00727628212
Bol0443062896B57, 80, 79, 90, 5961, 77Bra01466822912
Bol0444132781258, 96, 95, 83, 6065, 84Bra01695326712

6Bol006077392119471Bra0362513942BAt4G026404172B

7Bol0067342705B94Bra03031015111

8Bol0067354255A86Bra0303124303A

9Bol0067364663A93, 5558Bra0303144603AAt2g212305255A
Bol0458783723A48, 9050Bra0311723763A

10Bol006975149119574Bra02785514912At1g5953014812

11Bol0072953341194, 60, 90, 79, 94, 9962, 92, 90, 81Bra00144333111At1g6864045211
Bol0103083311194, 62, 91, 81, 98, 9465, 94, 91, 81Bra00432944111At3g1225035511
Bol0240004421161, 97, 61, 58, 62, 6187, 55, 62, 59Bra00924131011At5g0695033011
Bol0245263261180, 58, 81, 99, 81, 8060, 80, 81, 87Bra02871332611At5g069603301A
Bol0354523311199, 61, 92, 80, 95, 9464, 93, 90, 79Bra03476733111
Bol0439022461187, 57, 89, 76, 87, 8659, 83, 89, 77Bra03870533411

12Bol0080403801167, 80, 68, 86, 57, 7373, 77Bra00906336412At5g1003036412
Bol0092113671278, 98, 80, 91, 59, 8281, 89Bra02436636712At5g6521036812
Bol0190523901275, 89, 77, 87, 74, 9578, 86Bra02860436212
Bol0246363621288, 80, 99, 79, 50, 7189, 78Bra03187137012
Bol0437073641297, 77, 87, 76, 46, 6885, 75Bra03737431411
Bra03780939212

13Bol0080712011194, 54Bra03184513612
Bra02442424911

14Bol0082402331158, 90, 75, 6062Bra0154713921AAt1g060704231A
Bol0233333911A73, 62, 56, 8274Bra0182503741A
Bol0410353421A95, 62, 50, 7377Bra02173533911
Bra0306373811A

15Bol0088301021277Bra00597116011

16Bol009156188119376Bra03346420311At3g5196022812

17Bol0097133834A85, 9788Bra0163893684AAt1g2207038411
Bra0313643784A

18Bol0103901981194, 83, 8387Bra01971519311At1g1360019611
Bol0314411951178, 98, 9884Bra02689519511
Bra02689619511

19Bol0108361341298, 8178Bra00350013412At3g6242014612
Bol0445981411281, 9888Bra00767914112
Bol0331321711197, 8882, 56Bra02073517111At3g3053017311
Bol0430531721285, 9888, 59Bra02541817211At5g3880016512

20Bol0114703633A9569Bra0373823673AAt4g011203603A

21Bol0116839612
Bol03773310612

22Bol0117194326A92, 8378Bra0052874386AAt2g362704426A
Bra0172513966A

23Bol0121421606A61, 9561Bra0037551796AAt1g7539017311
Bol0393241606ABra0081921656A
Bol0398951786A94, 6276

24Bol0124721705B97, 85, 8479Bra0244781555BAt2g181601715B
Bol0414881695B86, 85, 9683Bra0372351685A
Bra0396311685A

25Bol0127032361295, 7273Bra03729023912At2g1677024912
Bol0426862441280, 8971Bra01304823912

26Bol0137122651187, 6361Bra01158023112At4g350402614B
Bol0346452551164, 9876Bra03466825511

27Bol0128552946A8854Bra03371926611At5g440803155B

28Bol0136234161A8975Bra0114854391AAt4g340004541A
Bol033853410117155

29Bol0136801541198, 89, 8681Bra0115451795BAt4g345901595B
Bol0242371485B89, 97, 7882Bra0176641535B
Bol0346761421184, 82, 9878Bra03463914211

30Bol0140511713A66, 54, 6668, 64Bra0053354223AAt1g321503893A
Bol0222594223A51, 50, 8570, 62Bra0230124033AAt2g355304093A
Bol0274513923A96, 66, 6463, 83Bra0232433523A
Bol0397994003A62, 85, 5558, 73

31Bol0152393911287Bra0336494141A

32Bol0160523941272Bra0107224454B

33Bol0162883741196, 7472Bra0278853731AAt1g5811037411
Bra0354641761B

34Bol0164322891179, 9884Bra00979329111At5g248002775B
Bol0223972871193, 7781Bra02047128911

35Bol016607142129478Bra01003514212At5g4945014511
Bol032354139128076

36Bol0036143531A80, 50, 7054, 72Bra0017423551BAt1g497204031A
Bol0167883071B52, 87, 5261, 48Bra0188003681BAt3g192904321A
Bol0180821331283, 64, 9659, 91Bra0375333881A
Bol0310023911A74, 57, 8853, 74

37Bol0170681871293, 83, 7360Bra01300518212At5g6083020612
Bol0362592101273, 85, 9560Bra02935310412
Bra03595718412

38Bol0185214421A7557Bra03358244611At4g389005531A

39Bol0185962431B69, 9570Bra0117802461BAt4g3773030511
Bol0288942461B94, 6672Bra0178502401B

40Bol0186882811173, 51, 9267Bra01050422211At4g359002855A
Bol0290422705B70, 66, 6462Bra0116482625A
Bol0299392651190, 59, 6666Bra0177352595B

41Bol020032891178, 76, 10082Bra01735917411At2g0403816611
Bol0325751761191, 81, 7869Bra0251441705B
Bol0427291705B80, 97, 7877Bra0265238911

42Bol0203903891188Bra00010236611

43Bol0212551944B77, 73, 9779Bra0063241814AAt5g158301864A
Bol0343711784A93, 71, 8275Bra0086701834B
Bol0304871874B70, 93, 7573Bra0235401884B

44Bol021964190126464Bra03602519012At3g4976015612
Bol037334186129364

45Bol0229251485B97, 84, 8892Bra0016711505BAt3g176091495B
Bol0308651455B86, 97, 8488Bra0212581465B
Bol0386601501181, 78, 9683Bra02222511612

46Bol0231616241091, 8759Bra02322459310At3g1080067510
Bra03414762910

47Bol0233563185A96, 8480Bra0306633205AAt1g068503375A
Bra0315413245A

48Bol0247041625B85, 9484Bra0089761645AAt5g112601685B
Bol0435891645B90, 8887Bra0233171665A

49Bol026864459119774Bra02574346211At1g1949047111

50Bol027526791129783Bra01564633912At1g779203681B

51Bol0277323716A67Bra0158473586A

52Bol028631120119773Bra03834112012At1g6888013812

53Bol0289753133A8782Bra0105723133AAt4g367303153A
96Bra0117013133A

54Bol033486303116555Bra0349252332BAt1g4299029510

55Bol0334892501184, 9670Bra0321913303AAt1g4370034111
Bol0432463303A99, 8269Bra03491626311

56Bol0334933101B9771Bra0349132221BAt1g354903001A

57Bol03780326611

58Bol0408592665A9364Bra0152812685AAt1g039702705A

59Bol041278333119380Bra01943633611At3g4446012

60Bol0438591491297Bra00928814712

61Bol0442924641067Bra01468043810

62Bol0451903854A8751Bra04026036411At1g452494271A

63Bol0458773863A85Bra0311733873A

Length: amino acid length of bZIP proteins. Identity 1: homology between B. oleracea and B. rapa. Identity 2: homology between B. oleracea and A. thaliana.

Group Classification domain bZIP number in B. oleraceabZIP number in B. rapa (Hwang et al.)bZIP number in A. thaliana (Hwang et al.)

Group 1 Q-rich domain131610
Group 2 D-rich domain 043
Group 3 P-rich domain 12126
Group 4 N-rich domain 594
Group 5 S-rich domain 131814
Group 6 G-rich domain 762
Group 10 Transmembrane domain 444
Group 11 Several LCRs 384117
Group 12 No LCR or UARR272613
Total 11913673

See reference [16].
3.3. Candidate BolbZIP Genes for Responses to Cold Stress

To identify BolbZIP genes that might function in responses to cold stress, we carried out comparative analysis of the expression of BolbZIP gene in two B. oleracea inbred lines, cold-tolerant BN106 and cold-susceptible BN107. BolbZIP genes were selected from an RNA sequencing dataset based on their annotations and their expression profiles were analyzed (data not shown). Among the 119 BolbZIP genes, the expression of 41 genes was remarkably changed in responses to cold treatment, whereas 78 genes of them showed no significant changes in their expression. BolbZIP genes with significantly different expression were determined in 4°C-treated sample base on fold change (FC) ≥2 and ≤0.5 relative to 22°C-treated sample. Cold treatment at this temperature caused the upregulation of 18 genes in BN106 and of 7 genes in BN107, whereas 15 genes were downregulated in BN106 and 8 genes were in BN107 by cold treatment. In total, the expression of 21 genes was upregulated and 20 genes downregulated by cold treatment (Table 3). In addition, 6 genes were not showing any expression within BN106 lines and therefore not calculated (Table 3). Finally, 47 BolbZIP genes’ expression level was confirmed using quantitative real-time PCR (qRT-PCR) (Table 3). To obtain detailed expression for the putative cold-response BolbZIP genes thus identified, qRT-PCR was carried out using samples from plants treated at several temperatures (22°C, 4°C, 0°C, or −2°C). Totally, 25 BolbZIP genes with significantly different expression were selected based on fold-changes (FC) ≥3 and ≤0.5 relative to the control sample (22°C). Most of the tested genes were significantly upregulated by cold treatment except Bol021255. Among 25 tested genes, 22 genes are displayed in Figure 2 and three genes by RT-PCR in Figure 3. We were not able to determine the analogous relative expression for the latter three genes because they were not expressed in the 22°C treated sample. The expression levels of several BolbZIP genes were comparable between the two lines. However, no significant change in the expression of Bol008071, Bol033132, and Bol042729 was observed in response to cold treatment in BN106, whereas these genes were upregulated at all temperatures in BN107 (Figure 2(a)). By contrast, Bol009713, Bol013712, Bol016432, and Bol022925 were upregulated in BN106, but not in BN107 (Figure 2(b)). The increased expression of 17 BolbZIP genes was more pronounced after severe cold treatment at 4°C, 0°C, and −2°C (Figure 2(c)) and one gene was downregulated by cold treatment in both BN106 and BN107 (Figure 2(d)). Homologs of cold stress-response BrbZIP genes were included in the qRT-PCR [16]. These expression patterns are summarized in Figure 4. Moreover, several genes including Bol016432, Bol022925, Bol026864, Bol027732, and Bol028975 displayed differential expression between cold (4°C) and freezing (−2°C) temperature. The expression level of the 3 genes, Bol008071, Bol033132, and Bol042729, was significantly increased in BN107 under cold conditions and was unchanged in BN106. Among three genes, Bol033132 has 97% sequence similarity to Bra020735 which was previously reported gene. Two proteins, Bol033132 and Bra020735, contained N-rich regions in LCRs (Figure 5(a)). Moreover, Bol042729 included the N-containing LCR (Figure 5(b)). We suggest the possibility that BolbZIP proteins as well as BrbZIP proteins containing N-rich regions might be involved in cold stress response.


Locus_IDFC1FC2Contigs
length (bp)
BRAD
Bol number
CDS
length (bp)
A. thaliana
homologs
Published
name
(BN106) value(BN107) value

Locus_018822.18 ± 0.110.0031 1.48 ± 0.020.0053 1948Bol0097131152AT1G22070
Locus_019090.16 ± 0.010.0052 0.65 ± 0.050.0105 1555Bol001886921AT2G42380
Locus_043583.84 ± 0.020.0002 NCNC1583Bol044598426AT3G62420
Locus_050134.58 ± 0.210.0158 1.89 ± 0.510.0044 1207Bol012472513AT2G18160GBF5
Locus_062922.35 ± 0.050.0044 1.20 ± 0.030.0474 1081Bol013712798AT4G35040
Locus_0886013.09 ± 0.320.0006 2.08 ± 0.110.0002 1541Bol0275262376AT1G77920TGA7
Locus_107232.99 ± 0.190.0012 2.28 ± 0.350.0077 1579Bol0268641380AT1G19490
Locus_109860.06 ± 0.000.0062 0.76 ± 0.040.0017 1117Bol016607429AT5G49450
Locus_110580.57 ± 0.030.0177 0.60 ± 0.040.0497 1354Bol004832903AT2G42380
Locus_113300.27 ± 0.010.0133 1.51 ± 0.330.0628 775Bol042729513AT2G04038
Locus_125590.35 ± 0.010.0090 0.87 ± 0.070.0994 1451Bol028975942AT4G36730GBF1
Locus_146430.83 ± 0.190.2500 0.32 ± 0.100.0346 816Bol033132516AT3G30530
Locus_147804.80 ± 0.930.0083 0.78 ± 0.030.0182 1882Bol014051516AT1G32150
Locus_150530.15 ± 0.000.0049 0.47 ± 0.030.0260 1757Bol0114701092AT4G01120GBF2
Locus_160594.67 ± 2.830.0358 1.29 ± 0.280.3559 873Bol0277321116
Locus_182581.46 ± 0.810.3124 NCNC1013Bol0117191299AT2G36270ABI5
Locus_192840.48 ± 0.020.0023 1.00 ± 0.160.0535 1580Bol0060771179AT4G02640BZO2H1
Locus_199755.14 ± 0.020.0015 3.11 ± 0.240.0000 1113Bol028894741AT4G37730
Locus_200382.25 ± 0.040.0002 0.74 ± 0.090.0017 2250Bol0338531233AT4G34000ABF3
Locus_214552.15 ± 0.030.0012 1.32 ± 0.050.0174 1248Bol041488510AT2G18160GBF5
Locus_222022.90 ± 0.220.0078 0.67 ± 0.030.0061 1566Bol000879936AT2G46270GBF3
Locus_229290.27 ± 0.050.0569 0.58 ± 0.110.0236 890Bol037803801
Locus_255347.11 ± 1.400.0024 1.87 ± 0.190.0400 1165Bol039895537AT1G75390
Locus_271200.13 ± 0.160.0645 NCNC545Bol008071606
Locus_28516NCNCNCNC284Bol033493933AT1G35490
Locus_315520.29 ± 0.050.0628 NCNC329Bol006902720AT2G41070DPBF4
Locus_318706.75 ± 3.180.0743 0.51 ± 0.170.1589 386Bol037733321
Locus_352740.57 ± 0.020.0027 0.19 ± 0.220.0014 1107Bol016432870AT5G24800BZO2H2
Locus_353360.12 ± 0.000.0113 0.17 ± 0.040.0006 969Bol021255585AT5G15830
Locus_359824.94 ± 0.070.0010 3.61 ± 0.300.0016 1216Bol034676429AT4G34590ATB2/GBF6
Locus_366440.40 ± 0.060.0362 0.70 ± 0.070.1179 1588Bol0080401143AT5G65210TGA1
Locus_382070.56 ± 0.060.0396 0.18 ± 0.210.0272 673Bol0051151032AT2G40620
Locus_383001.23 ± 0.640.5000 0.00 NC318Bol018596732AT4G37730
Locus_385334.51 ± 0.340.0023 0.85 ± 0.090.0454 1978Bol0437071095AT5G10030TGA4
Locus_386369.75 ± 0.870.0272 0.56 ± 0.050.0346 487Bol030865438AT3G17609HYH
Locus_391771.20 ± 0.180.1749 0.38 ± 0.010.0054 839Bol043589495AT5G11260HY5
Locus_398370.78 ± 0.050.0267 2.20 ± 1.210.0097 1648Bol0410351038AT1G06070
Locus_39980NCNCNCNC478Bol038660453AT3G17609HYH
Locus_410800.07 ± 0.040.0033 2.32 ± 0.800.0840 677Bol010390597AT1G13600
Locus_44632NCNCNCNC256Bol029939798AT4G35900FD-1
Locus_449502.86 ± 0.000.0052 1.32 ± 0.050.0301 1447Bol024526981AT5G06960TGA5/OBF5
Locus_45018NCNC0.70 ± 0.020.0223 667Bol022925447AT3G17609HYH
Locus_469510.15 ± 0.170.0257 NCNC462Bol020032270AT2G04038
Locus_47897NCNCNCNC458Bol037334561AT3G49760
Locus_490750.39 ± 0.100.0149 2.76 ± 0.400.0145 739Bol034371537AT5G15830
Locus_55049NCNC0.57 ± 0.290.1464 311Bol018688846AT4G35900FD-1
Locus_560350.04 ± 0.000.0010 0.15 ± 0.060.0055 662Bol032354420AT5G49450

NC, not calculated. FC1, signal intensity of 0°C treated plant over control plant (22°C) in BN106. FC2, signal intensity of 0°C treated plant over control plant in BN107.

4. Discussion

It was known that B. rapa and B. oleracea genomes are highly similar in their gene structure, but there still exist species-specific genes in two species. Hence this study was carried out in B. oleracea and identified 119 BolbZIP proteins and placed them into 63 categories according to sequence similarity (Table 1). To identify the bZIP proteins in B. oleracea, a few bZIP domain consensus sequences of several species were used (Table S2). It is possible that this approach could lead us to underestimate the number of bZIP proteins present, despite the high number of BolbZIP proteins we identified. To address this, other search methods or more detailed consensus sequences for bZIP proteins in plants could be examined. In Arabidopsis, bZIP proteins were classified into different groups and subfamilies according to sequence similarities in their basic region and additional conserved motifs in order to elucidate the likely function of the proteins [21]. In rice, Nijhawan et al. [19] published 89 bZIP transcription factor-encoding genes based on DNA binding specificity and amino acid sequences in basic and hinge regions. Recently BrbZIP and AtbZIP proteins were divided into 9 groups based on their UARR and LCRs, which are highly enriched in one or a few amino acids [16]. In this study, 119 BolbZIP proteins were categorized into 63 groups and also classified according to UARR and LCRs based on the classification method of Hwang et al. [16]. In addition, the sequence similarity of the bZIP proteins of B. oleracea, B. rapa, and A. thaliana was analyzed. Most of homologs were found to have the same UARR and LCRs. UARRs were composed of 6 amino acids including Q, D, P, N, S, and G in the B. oleracea (Tables 2 and S4). This conservation of amino acid composition suggests that these 6 amino acids are important for biological functions and formation of protein structures in bZIP proteins.

BolbZIP gene family members were physically mapped to all the nine chromosomes of B. oleracea. Among them, chromosome 04 was found to contain the highest number of BolbZIP genes (21%), while chromosomes 05 and 06 harbored the fewest (6-7%) (Figure 1, Table S3). In B. rapa, the highest number of BrbZIP genes was detected in chromosome 09 (21%) [16]. Additionally, most BolbZIP genes were distributed in the arm end of each chromosome. The clustered distribution pattern of the BolbZIP genes on some chromosomes might be indicated in significant regions evolutionarily. For example, BolbZIP genes located on chromosomes 01, 02, 04, 07, and 08, and chromosomes 09 appear to be clustered at the arm end in those chromosomes (Figure 1).

To screen for cold stress-responsive BolbZIP genes, we tested the transcription patterns of BolbZIP genes enhanced or decreased by cold treatment in two B. oleracea lines that showed different cold tolerance [16]. Based on their expression patterns, the cold-responsive BolbZIP transcription factors were divided into four groups (Figure 2). We found that the expression of three genes, Bol008071, Bol033132, and Bol042729, was upregulated in cold-susceptible BN107 but not changed in cold-tolerant BN106. Additionally, when compared with 6 genes published for significant BrbZIP factors involved in the cold response, 4 BolbZIP genes (Bol004832, homologous to Bra000256, Bra004689, and Bra003320; Bol033132, homologous to Bra020735; Bol018688, homologous to Bra011648; and Bol021255, homologous to Bra023540) showed similar patterns of expression in response to cold treatment. For example, Bol033132 showed an expression pattern like that of its homolog Bra020735, indicating that these genes might be conserved key regulator in cold stress responses. Moreover, Bol033132 and Bol042729 encode bZIP proteins that include the LCR containing amino acid N or N-rich region (Figure 5, Tables S4 and S5). These results indicated that the N-containing region of BolbZIP proteins might be involved in cold stress responses. Although the functions of the N-containing region are largely unknown, the regions might be biologically active [24, 25]. This genome-wide identification and expression profiling of bZIP proteins from B. oleracea provides new opportunities for functional analyses, which may be used in further studies for improving stress tolerance in plants.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

This research was supported by Golden Seed Project (Center for Horticultural Seed Development, no. 213003-04-4-SB110), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA), and Korea Forest Service (KFS).

Supplementary Materials

The supplementary materials contain 5 files, they are some important data supported to the methods and results of the presented study. These data make paper easier to read and understand.

  1. Supplementary Materials

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Copyright © 2016 Indeok Hwang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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