# Genome-Wide Identification and Characterization of bZIP Transcription Factors inBrassica oleracea under Cold Stress

Authors: Indeok Hwang; Ranjith Kumar Manoharan; Jong-Goo Kang; Mi-Young Chung; Young-Wook Kim; Ill-Sup Nou
Journal: BioMed Research International
 (2016)

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## 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-responsiveBolbZIP genes were screened in inbred lines,BN106 andBN107, using RNA sequencing data and qRT-PCR. The expression level of the 3 genes,Bol008071,Bol033132, andBol042729, was significantly increased inBN107 under cold conditions and was unchanged inBN106. The upregulation of these genes inBN107, 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 toBra020735, which was identified in a screen for cold-related genes inB. 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.


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## Body

## 1. Introduction

Cabbage (Brassica oleracea L.) plants represent one of the major vegetable crops grown worldwide. Most crops ofB. oleracea and its sister speciesBrassica 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 ofB. oleracea (with the CC genome) andB. rapa (with the AA genome) were recently published [3, 4]. A total of 66.5% (34,237) ofB. oleracea genes and 74.9% (34,324) ofB. rapa genes were clustered. In total, 5,735B. rapa-specific genes and 9,832B. 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 inB. 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 inArabidopsis according to sequence similarities of their basic region and additional conserved motifs, and 141 inHordeum vulgare [16–22]. However, little is known about the genome-wide survey and expression patterns of bZIP transcription factors inB. oleracea. Among the BolbZIPs, the function of only one gene related with drought stress and ABA has been reported. Expression ofBolABI5 was dramatically induced by drought stress and exogenous ABA [23]. Heterogeneous expression ofBolABI5 rescued the ABA-insensitive phenotype of theArabidopsis abi5-1 mutant during seed germination, suggesting thatBolABI5 likely functions in positive regulation of plant ABA responses.The bZIP domain includes a basic region and a leucine zipper located on a contiguousα-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 inB. oleracea.


## 2. Materials and Methods

### 2.1. Database Searches for bZIP Transcription Factors inB. 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 (E-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 ofB. 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 5μL (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 andBN107 which exhibit different sensitivity to cold stress, were used for RNA sequencing. Total RNA was extracted from leaves ofBN106 andBN107 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 E-value ≤ 1
e
-
10). 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 theBolbZIP 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. Anactin primer set forB. oleracea was used for normalization of RT-PCR and qRT-PCR.


## 2.1. Database Searches for bZIP Transcription Factors inB. 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 (E-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 ofB. 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 5μL (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 andBN107 which exhibit different sensitivity to cold stress, were used for RNA sequencing. Total RNA was extracted from leaves ofBN106 andBN107 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 E-value ≤ 1
e
-
10). 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 theBolbZIP 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. Anactin primer set forB. oleracea was used for normalization of RT-PCR and qRT-PCR.


## 3. Results

### 3.1. Identification of bZIP Transcription Factors inB. oleracea

To search for bZIP transcription factors inB. oleracea, we used the conserved bZIP domain consensus sequences (Table S2) of several proteins as BLASTP queries against theBrassica 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 E-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 candidateBolbZIP gene inB. oleracea chromosome data available at Bolbase (Version 1.0) was then determined.Among 119 candidateBolbZIP genes, 112 were mapped on chromosomes C01–C09 (Figure 1). 14 genes ofBolbZIP 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 theBolbZIP 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, andBol000879 on Scaffold000492.Figure 1
Distribution ofBolbZIP genes onto the nine assembledB. oleracea chromosomes. Graphical (scaled) representation of physical locations for eachBolbZIP gene onB. oleracea chromosomes (numbered C01–C09). Chromosomal distances are given in Mbp.


### 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 (Table1) [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 fromB. rapa andA. 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.Table 1
119 BolbZIP proteins were divided into 63 categories based on amino acid sequence similarity.
Index
B. oleracea
Identity 1 (%)
Identity 2 (%)
B. rapa homologs
A. thaliana homologs
Bol number
Length (aa)
Group
Bra number
Length (aa)
Group
At number
Length (aa)
Group
1
Bol000879
311
3A
95
75
Bra004550
379
3A
At2g46270
382
3A
Bol017742
328
3A
80
70
Bol029580
300
11
76
79
2
Bol004832
300
11
65, 98, 62, 62
75, 64
Bra000256
362
11
At2g42380
321
4A
Bol001886
306
11
82, 75, 61, 62
71, 65
Bra004689
306
4B
At3g58120
329
4A
Bra007380
318
4A
Bra003320
304
11
3
Bol005115
343
1A
62, 83,92
83
Bra000195
334
1A
At2g40620
367
1A
Bol006882
356
1A
59, 98, 86
79
Bra004582
356
1A
Bol020604
336
1A
88, 66, 67
66
Bra016980
342
1A
4
Bol005139
617
10
79
53
Bra016959
624
10
At2g40950
721
10
Bol006897
639
10
65
61
5
Bol004200
281
12
59, 83, 84, 83, 60
64, 88
Bra004597
281
12
At2g41070
262
12
Bol005146
272
12
74, 59, 60, 60, 93
69, 60
Bra007274
282
12
At3g56850
297
6B
Bol006902
239
12
93, 59, 60, 61, 70
64, 58
Bra007276
282
12
Bol044306
289
6B
57, 80, 79, 90, 59
61, 77
Bra014668
229
12
Bol044413
278
12
58, 96, 95, 83, 60
65, 84
Bra016953
267
12
6
Bol006077
392
11
94
71
Bra036251
394
2B
At4G02640
417
2B
7
Bol006734
270
5B
94
Bra030310
151
11
8
Bol006735
425
5A
86
Bra030312
430
3A
9
Bol006736
466
3A
93, 55
58
Bra030314
460
3A
At2g21230
525
5A
Bol045878
372
3A
48, 90
50
Bra031172
376
3A
10
Bol006975
149
11
95
74
Bra027855
149
12
At1g59530
148
12
11
Bol007295
334
11
94, 60, 90, 79, 94, 99
62, 92, 90, 81
Bra001443
331
11
At1g68640
452
11
Bol010308
331
11
94, 62, 91, 81, 98, 94
65, 94, 91, 81
Bra004329
441
11
At3g12250
355
11
Bol024000
442
11
61, 97, 61, 58, 62, 61
87, 55, 62, 59
Bra009241
310
11
At5g06950
330
11
Bol024526
326
11
80, 58, 81, 99, 81, 80
60, 80, 81, 87
Bra028713
326
11
At5g06960
330
1A
Bol035452
331
11
99, 61, 92, 80, 95, 94
64, 93, 90, 79
Bra034767
331
11
Bol043902
246
11
87, 57, 89, 76, 87, 86
59, 83, 89, 77
Bra038705
334
11
12
Bol008040
380
11
67, 80, 68, 86, 57, 73
73, 77
Bra009063
364
12
At5g10030
364
12
Bol009211
367
12
78, 98, 80, 91, 59, 82
81, 89
Bra024366
367
12
At5g65210
368
12
Bol019052
390
12
75, 89, 77, 87, 74, 95
78, 86
Bra028604
362
12
Bol024636
362
12
88, 80, 99, 79, 50, 71
89, 78
Bra031871
370
12
Bol043707
364
12
97, 77, 87, 76, 46, 68
85, 75
Bra037374
314
11
Bra037809
392
12
13
Bol008071
201
11
94, 54
Bra031845
136
12
Bra024424
249
11
14
Bol008240
233
11
58, 90, 75, 60
62
Bra015471
392
1A
At1g06070
423
1A
Bol023333
391
1A
73, 62, 56, 82
74
Bra018250
374
1A
Bol041035
342
1A
95, 62, 50, 73
77
Bra021735
339
11
Bra030637
381
1A
15
Bol008830
102
12
77
Bra005971
160
11
16
Bol009156
188
11
93
76
Bra033464
203
11
At3g51960
228
12
17
Bol009713
383
4A
85, 97
88
Bra016389
368
4A
At1g22070
384
11
Bra031364
378
4A
18
Bol010390
198
11
94, 83, 83
87
Bra019715
193
11
At1g13600
196
11
Bol031441
195
11
78, 98, 98
84
Bra026895
195
11
Bra026896
195
11
19
Bol010836
134
12
98, 81
78
Bra003500
134
12
At3g62420
146
12
Bol044598
141
12
81, 98
88
Bra007679
141
12
Bol033132
171
11
97, 88
82, 56
Bra020735
171
11
At3g30530
173
11
Bol043053
172
12
85, 98
88, 59
Bra025418
172
11
At5g38800
165
12
20
Bol011470
363
3A
95
69
Bra037382
367
3A
At4g01120
360
3A
21
Bol011683
96
12
Bol037733
106
12
22
Bol011719
432
6A
92, 83
78
Bra005287
438
6A
At2g36270
442
6A
Bra017251
396
6A
23
Bol012142
160
6A
61, 95
61
Bra003755
179
6A
At1g75390
173
11
Bol039324
160
6A
Bra008192
165
6A
Bol039895
178
6A
94, 62
76
24
Bol012472
170
5B
97, 85, 84
79
Bra024478
155
5B
At2g18160
171
5B
Bol041488
169
5B
86, 85, 96
83
Bra037235
168
5A
Bra039631
168
5A
25
Bol012703
236
12
95, 72
73
Bra037290
239
12
At2g16770
249
12
Bol042686
244
12
80, 89
71
Bra013048
239
12
26
Bol013712
265
11
87, 63
61
Bra011580
231
12
At4g35040
261
4B
Bol034645
255
11
64, 98
76
Bra034668
255
11
27
Bol012855
294
6A
88
54
Bra033719
266
11
At5g44080
315
5B
28
Bol013623
416
1A
89
75
Bra011485
439
1A
At4g34000
454
1A
Bol033853
410
11
71
55
29
Bol013680
154
11
98, 89, 86
81
Bra011545
179
5B
At4g34590
159
5B
Bol024237
148
5B
89, 97, 78
82
Bra017664
153
5B
Bol034676
142
11
84, 82, 98
78
Bra034639
142
11
30
Bol014051
171
3A
66, 54, 66
68, 64
Bra005335
422
3A
At1g32150
389
3A
Bol022259
422
3A
51, 50, 85
70, 62
Bra023012
403
3A
At2g35530
409
3A
Bol027451
392
3A
96, 66, 64
63, 83
Bra023243
352
3A
Bol039799
400
3A
62, 85, 55
58, 73
31
Bol015239
391
12
87
Bra033649
414
1A
32
Bol016052
394
12
72
Bra010722
445
4B
33
Bol016288
374
11
96, 74
72
Bra027885
373
1A
At1g58110
374
11
Bra035464
176
1B
34
Bol016432
289
11
79, 98
84
Bra009793
291
11
At5g24800
277
5B
Bol022397
287
11
93, 77
81
Bra020471
289
11
35
Bol016607
142
12
94
78
Bra010035
142
12
At5g49450
145
11
Bol032354
139
12
80
76
36
Bol003614
353
1A
80, 50, 70
54, 72
Bra001742
355
1B
At1g49720
403
1A
Bol016788
307
1B
52, 87, 52
61, 48
Bra018800
368
1B
At3g19290
432
1A
Bol018082
133
12
83, 64, 96
59, 91
Bra037533
388
1A
Bol031002
391
1A
74, 57, 88
53, 74
37
Bol017068
187
12
93, 83, 73
60
Bra013005
182
12
At5g60830
206
12
Bol036259
210
12
73, 85, 95
60
Bra029353
104
12
Bra035957
184
12
38
Bol018521
442
1A
75
57
Bra033582
446
11
At4g38900
553
1A
39
Bol018596
243
1B
69, 95
70
Bra011780
246
1B
At4g37730
305
11
Bol028894
246
1B
94, 66
72
Bra017850
240
1B
40
Bol018688
281
11
73, 51, 92
67
Bra010504
222
11
At4g35900
285
5A
Bol029042
270
5B
70, 66, 64
62
Bra011648
262
5A
Bol029939
265
11
90, 59, 66
66
Bra017735
259
5B
41
Bol020032
89
11
78, 76, 100
82
Bra017359
174
11
At2g04038
166
11
Bol032575
176
11
91, 81, 78
69
Bra025144
170
5B
Bol042729
170
5B
80, 97, 78
77
Bra026523
89
11
42
Bol020390
389
11
88
Bra000102
366
11
43
Bol021255
194
4B
77, 73, 97
79
Bra006324
181
4A
At5g15830
186
4A
Bol034371
178
4A
93, 71, 82
75
Bra008670
183
4B
Bol030487
187
4B
70, 93, 75
73
Bra023540
188
4B
44
Bol021964
190
12
64
64
Bra036025
190
12
At3g49760
156
12
Bol037334
186
12
93
64
45
Bol022925
148
5B
97, 84, 88
92
Bra001671
150
5B
At3g17609
149
5B
Bol030865
145
5B
86, 97, 84
88
Bra021258
146
5B
Bol038660
150
11
81, 78, 96
83
Bra022225
116
12
46
Bol023161
624
10
91, 87
59
Bra023224
593
10
At3g10800
675
10
Bra034147
629
10
47
Bol023356
318
5A
96, 84
80
Bra030663
320
5A
At1g06850
337
5A
Bra031541
324
5A
48
Bol024704
162
5B
85, 94
84
Bra008976
164
5A
At5g11260
168
5B
Bol043589
164
5B
90, 88
87
Bra023317
166
5A
49
Bol026864
459
11
97
74
Bra025743
462
11
At1g19490
471
11
50
Bol027526
791
12
97
83
Bra015646
339
12
At1g77920
368
1B
51
Bol027732
371
6A
67
Bra015847
358
6A
52
Bol028631
120
11
97
73
Bra038341
120
12
At1g68880
138
12
53
Bol028975
313
3A
87
82
Bra010572
313
3A
At4g36730
315
3A
96
Bra011701
313
3A
54
Bol033486
303
11
65
55
Bra034925
233
2B
At1g42990
295
10
55
Bol033489
250
11
84, 96
70
Bra032191
330
3A
At1g43700
341
11
Bol043246
330
3A
99, 82
69
Bra034916
263
11
56
Bol033493
310
1B
97
71
Bra034913
222
1B
At1g35490
300
1A
57
Bol037803
266
11
58
Bol040859
266
5A
93
64
Bra015281
268
5A
At1g03970
270
5A
59
Bol041278
333
11
93
80
Bra019436
336
11
At3g44460
12
60
Bol043859
149
12
97
Bra009288
147
12
61
Bol044292
464
10
67
Bra014680
438
10
62
Bol045190
385
4A
87
51
Bra040260
364
11
At1g45249
427
1A
63
Bol045877
386
3A
85
Bra031173
387
3A
Length: amino acid length of bZIP proteins. Identity 1: homology betweenB. oleracea and B. rapa. Identity 2: homology between B. oleracea and A. thaliana.Table 2
Number of bZIP transcription factors in each group based on UARR and LCRs.
Group
Classification domain
bZIP number inB. oleracea
bZIP number inB. rapa (Hwang et al.∗)
bZIP number inA. thaliana (Hwang et al.∗)
Group 1
Q-rich domain
13
16
10
Group 2
D-rich domain
0
4
3
Group 3
P-rich domain
12
12
6
Group 4
N-rich domain
5
9
4
Group 5
S-rich domain
13
18
14
Group 6
G-rich domain
7
6
2
Group 10
Transmembrane domain
4
4
4
Group 11
Several LCRs
38
41
17
Group 12
No LCR or UARR
27
26
13
Total
—
119
136
73
∗See reference [16].


### 3.3. CandidateBolbZIP 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 ofBolbZIP gene in twoB. oleracea inbred lines, cold-tolerantBN106 and cold-susceptibleBN107.BolbZIP genes were selected from an RNA sequencing dataset based on their annotations and their expression profiles were analyzed (data not shown). Among the 119BolbZIP 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 inBN106 and of 7 genes inBN107, whereas 15 genes were downregulated inBN106 and 8 genes were inBN107 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 withinBN106 lines and therefore not calculated (Table 3). Finally, 47BolbZIP 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, 25BolbZIP 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 exceptBol021255. 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 severalBolbZIP genes were comparable between the two lines. However, no significant change in the expression ofBol008071, Bol033132, and Bol042729 was observed in response to cold treatment inBN106, whereas these genes were upregulated at all temperatures inBN107 (Figure 2(a)). By contrast,Bol009713, Bol013712, Bol016432, and Bol022925 were upregulated inBN106, but not inBN107 (Figure 2(b)). The increased expression of17 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 bothBN106 andBN107 (Figure 2(d)). Homologs of cold stress-responseBrbZIP genes were included in the qRT-PCR [16]. These expression patterns are summarized in Figure 4. Moreover, several genes includingBol016432,Bol022925,Bol026864,Bol027732, andBol028975 displayed differential expression between cold (4°C) and freezing (−2°C) temperature. The expression level of the 3 genes,Bol008071,Bol033132, andBol042729, was significantly increased inBN107 under cold conditions and was unchanged inBN106. Among three genes,Bol033132 has 97% sequence similarity toBra020735 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.Table 3
Cold-treatment induced change in expression based on RNA sequencing data. The differentially expressed genes determined based on fold change (FC) ≥2 are displayed with bold font and ≤0.5 with italic font.
Locus_ID
FC1
FC2
Contigslength (bp)
BRADBol number
CDSlength (bp)
A. thaliana homologs
Publishedname
(BN106)
P value
(BN107)
P value
Locus_01882
2.18 ± 0.11
0.0031
1.48 ± 0.02
0.0053
1948
Bol009713
1152
AT1G22070
Locus_01909
0.16 ± 0.01
0.0052
0.65 ± 0.05
0.0105
1555
Bol001886
921
AT2G42380
Locus_04358
3.84 ± 0.02
0.0002
NC
NC
1583
Bol044598
426
AT3G62420
Locus_05013
4.58 ± 0.21
0.0158
1.89 ± 0.51
0.0044
1207
Bol012472
513
AT2G18160
GBF5
Locus_06292
2.35 ± 0.05
0.0044
1.20 ± 0.03
0.0474
1081
Bol013712
798
AT4G35040
Locus_08860
13.09 ± 0.32
0.0006
2.08 ± 0.11
0.0002
1541
Bol027526
2376
AT1G77920
TGA7
Locus_10723
2.99 ± 0.19
0.0012
2.28 ± 0.35
0.0077
1579
Bol026864
1380
AT1G19490
Locus_10986
0.06 ± 0.00
0.0062
0.76 ± 0.04
0.0017
1117
Bol016607
429
AT5G49450
Locus_11058
0.57 ± 0.03
0.0177
0.60 ± 0.04
0.0497
1354
Bol004832
903
AT2G42380
Locus_11330
0.27 ± 0.01
0.0133
1.51 ± 0.33
0.0628
775
Bol042729
513
AT2G04038
Locus_12559
0.35 ± 0.01
0.0090
0.87 ± 0.07
0.0994
1451
Bol028975
942
AT4G36730
GBF1
Locus_14643
0.83 ± 0.19
0.2500
0.32 ± 0.10
0.0346
816
Bol033132
516
AT3G30530
Locus_14780
4.80 ± 0.93
0.0083
0.78 ± 0.03
0.0182
1882
Bol014051
516
AT1G32150
Locus_15053
0.15 ± 0.00
0.0049
0.47 ± 0.03
0.0260
1757
Bol011470
1092
AT4G01120
GBF2
Locus_16059
4.67 ± 2.83
0.0358
1.29 ± 0.28
0.3559
873
Bol027732
1116
Locus_18258
1.46 ± 0.81
0.3124
NC
NC
1013
Bol011719
1299
AT2G36270
ABI5
Locus_19284
0.48 ± 0.02
0.0023
1.00 ± 0.16
0.0535
1580
Bol006077
1179
AT4G02640
BZO2H1
Locus_19975
5.14 ± 0.02
0.0015
3.11 ± 0.24
0.0000
1113
Bol028894
741
AT4G37730
Locus_20038
2.25 ± 0.04
0.0002
0.74 ± 0.09
0.0017
2250
Bol033853
1233
AT4G34000
ABF3
Locus_21455
2.15 ± 0.03
0.0012
1.32 ± 0.05
0.0174
1248
Bol041488
510
AT2G18160
GBF5
Locus_22202
2.90 ± 0.22
0.0078
0.67 ± 0.03
0.0061
1566
Bol000879
936
AT2G46270
GBF3
Locus_22929
0.27 ± 0.05
0.0569
0.58 ± 0.11
0.0236
890
Bol037803
801
Locus_25534
7.11 ± 1.40
0.0024
1.87 ± 0.19
0.0400
1165
Bol039895
537
AT1G75390
Locus_27120
0.13 ± 0.16
0.0645
NC
NC
545
Bol008071
606
Locus_28516
NC
NC
NC
NC
284
Bol033493
933
AT1G35490
Locus_31552
0.29 ± 0.05
0.0628
NC
NC
329
Bol006902
720
AT2G41070
DPBF4
Locus_31870
6.75 ± 3.18
0.0743
0.51 ± 0.17
0.1589
386
Bol037733
321
Locus_35274
0.57 ± 0.02
0.0027
0.19 ± 0.22
0.0014
1107
Bol016432
870
AT5G24800
BZO2H2
Locus_35336
0.12 ± 0.00
0.0113
0.17 ± 0.04
0.0006
969
Bol021255
585
AT5G15830
Locus_35982
4.94 ± 0.07
0.0010
3.61 ± 0.30
0.0016
1216
Bol034676
429
AT4G34590
ATB2/GBF6
Locus_36644
0.40 ± 0.06
0.0362
0.70 ± 0.07
0.1179
1588
Bol008040
1143
AT5G65210
TGA1
Locus_38207
0.56 ± 0.06
0.0396
0.18 ± 0.21
0.0272
673
Bol005115
1032
AT2G40620
Locus_38300
1.23 ± 0.64
0.5000
0.00
NC
318
Bol018596
732
AT4G37730
Locus_38533
4.51 ± 0.34
0.0023
0.85 ± 0.09
0.0454
1978
Bol043707
1095
AT5G10030
TGA4
Locus_38636
9.75 ± 0.87
0.0272
0.56 ± 0.05
0.0346
487
Bol030865
438
AT3G17609
HYH
Locus_39177
1.20 ± 0.18
0.1749
0.38 ± 0.01
0.0054
839
Bol043589
495
AT5G11260
HY5
Locus_39837
0.78 ± 0.05
0.0267
2.20 ± 1.21
0.0097
1648
Bol041035
1038
AT1G06070
Locus_39980
NC
NC
NC
NC
478
Bol038660
453
AT3G17609
HYH
Locus_41080
0.07 ± 0.04
0.0033
2.32 ± 0.80
0.0840
677
Bol010390
597
AT1G13600
Locus_44632
NC
NC
NC
NC
256
Bol029939
798
AT4G35900
FD-1
Locus_44950
2.86 ± 0.00
0.0052
1.32 ± 0.05
0.0301
1447
Bol024526
981
AT5G06960
TGA5/OBF5
Locus_45018
NC
NC
0.70 ± 0.02
0.0223
667
Bol022925
447
AT3G17609
HYH
Locus_46951
0.15 ± 0.17
0.0257
NC
NC
462
Bol020032
270
AT2G04038
Locus_47897
NC
NC
NC
NC
458
Bol037334
561
AT3G49760
Locus_49075
0.39 ± 0.10
0.0149
2.76 ± 0.40
0.0145
739
Bol034371
537
AT5G15830
Locus_55049
NC
NC
0.57 ± 0.29
0.1464
311
Bol018688
846
AT4G35900
FD-1
Locus_56035
0.04 ± 0.00
0.0010
0.15 ± 0.06
0.0055
662
Bol032354
420
AT5G49450
NC, not calculated. FC1, signal intensity of 0°C treated plant over control plant (22°C) inBN106. FC2, signal intensity of 0°C treated plant over control plant in BN107.Figure 2
Relative expression levels of 22BolbZIP genes in cabbage inbred lines cold-tolerantBN106 and cold-susceptibleBN107 under cold stress conditions. 5-week-old plants were treated at 4°C, 0°C, and −2°C. Theactin transcript levels were used for normalization. Values shown are relative to transcript levels in the 22°C treated plants. Error bars indicate standard deviation. (a) Genes showing no significant relative expression change inBN106 and upregulating at all temperatures inBN107. (b) Genes showing upregulation at all temperatures inBN106 and no significant relative expression change inBN107. (c) Genes showing greater upregulation at lower temperatures inBN106 andBN107. (d) Genes showing downregulation in response to cold inBN106 andBN107.Figure 3
RT-PCR analysis of threeBolbZIP genes in response to cold. These genes showed no expression in 22°C-treated cabbage inbred linesBN106 andBN107. Theactin transcript levels were used as an internal control.Figure 4
Heat map representation of cold-responsive expression ofBolbZIP andBrbZIP genes. The expression pattern of the BolbZIPs and their closest BrbZIP homologs in response to cold (4°C) and freezing (−2°C) stresses are shown. Heat map was generated using up- and downregulated gene expression data from qRT-PCR and RT-PCR results.Figure 5
Amino acid sequences of Bol003312 and Bol042749 and their homologs. (a) An alignment of the amino acid sequences of Bol033132 and two homologs, Bra020735 and At3g30530. Conserved sequences of bZIP domain are highlighted using gray shade in the basic and leucine zipper regions. (b) An alignment of the amino acid sequences of Bol042729 and two homologs, Bra025144 and At2g04038.
(a)
(b)


## 3.1. Identification of bZIP Transcription Factors inB. oleracea

To search for bZIP transcription factors inB. oleracea, we used the conserved bZIP domain consensus sequences (Table S2) of several proteins as BLASTP queries against theBrassica 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 E-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 candidateBolbZIP gene inB. oleracea chromosome data available at Bolbase (Version 1.0) was then determined.Among 119 candidateBolbZIP genes, 112 were mapped on chromosomes C01–C09 (Figure 1). 14 genes ofBolbZIP 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 theBolbZIP 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, andBol000879 on Scaffold000492.Figure 1
Distribution ofBolbZIP genes onto the nine assembledB. oleracea chromosomes. Graphical (scaled) representation of physical locations for eachBolbZIP gene onB. oleracea chromosomes (numbered C01–C09). Chromosomal distances are given in Mbp.


## 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 (Table1) [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 fromB. rapa andA. 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.Table 1
119 BolbZIP proteins were divided into 63 categories based on amino acid sequence similarity.
Index
B. oleracea
Identity 1 (%)
Identity 2 (%)
B. rapa homologs
A. thaliana homologs
Bol number
Length (aa)
Group
Bra number
Length (aa)
Group
At number
Length (aa)
Group
1
Bol000879
311
3A
95
75
Bra004550
379
3A
At2g46270
382
3A
Bol017742
328
3A
80
70
Bol029580
300
11
76
79
2
Bol004832
300
11
65, 98, 62, 62
75, 64
Bra000256
362
11
At2g42380
321
4A
Bol001886
306
11
82, 75, 61, 62
71, 65
Bra004689
306
4B
At3g58120
329
4A
Bra007380
318
4A
Bra003320
304
11
3
Bol005115
343
1A
62, 83,92
83
Bra000195
334
1A
At2g40620
367
1A
Bol006882
356
1A
59, 98, 86
79
Bra004582
356
1A
Bol020604
336
1A
88, 66, 67
66
Bra016980
342
1A
4
Bol005139
617
10
79
53
Bra016959
624
10
At2g40950
721
10
Bol006897
639
10
65
61
5
Bol004200
281
12
59, 83, 84, 83, 60
64, 88
Bra004597
281
12
At2g41070
262
12
Bol005146
272
12
74, 59, 60, 60, 93
69, 60
Bra007274
282
12
At3g56850
297
6B
Bol006902
239
12
93, 59, 60, 61, 70
64, 58
Bra007276
282
12
Bol044306
289
6B
57, 80, 79, 90, 59
61, 77
Bra014668
229
12
Bol044413
278
12
58, 96, 95, 83, 60
65, 84
Bra016953
267
12
6
Bol006077
392
11
94
71
Bra036251
394
2B
At4G02640
417
2B
7
Bol006734
270
5B
94
Bra030310
151
11
8
Bol006735
425
5A
86
Bra030312
430
3A
9
Bol006736
466
3A
93, 55
58
Bra030314
460
3A
At2g21230
525
5A
Bol045878
372
3A
48, 90
50
Bra031172
376
3A
10
Bol006975
149
11
95
74
Bra027855
149
12
At1g59530
148
12
11
Bol007295
334
11
94, 60, 90, 79, 94, 99
62, 92, 90, 81
Bra001443
331
11
At1g68640
452
11
Bol010308
331
11
94, 62, 91, 81, 98, 94
65, 94, 91, 81
Bra004329
441
11
At3g12250
355
11
Bol024000
442
11
61, 97, 61, 58, 62, 61
87, 55, 62, 59
Bra009241
310
11
At5g06950
330
11
Bol024526
326
11
80, 58, 81, 99, 81, 80
60, 80, 81, 87
Bra028713
326
11
At5g06960
330
1A
Bol035452
331
11
99, 61, 92, 80, 95, 94
64, 93, 90, 79
Bra034767
331
11
Bol043902
246
11
87, 57, 89, 76, 87, 86
59, 83, 89, 77
Bra038705
334
11
12
Bol008040
380
11
67, 80, 68, 86, 57, 73
73, 77
Bra009063
364
12
At5g10030
364
12
Bol009211
367
12
78, 98, 80, 91, 59, 82
81, 89
Bra024366
367
12
At5g65210
368
12
Bol019052
390
12
75, 89, 77, 87, 74, 95
78, 86
Bra028604
362
12
Bol024636
362
12
88, 80, 99, 79, 50, 71
89, 78
Bra031871
370
12
Bol043707
364
12
97, 77, 87, 76, 46, 68
85, 75
Bra037374
314
11
Bra037809
392
12
13
Bol008071
201
11
94, 54
Bra031845
136
12
Bra024424
249
11
14
Bol008240
233
11
58, 90, 75, 60
62
Bra015471
392
1A
At1g06070
423
1A
Bol023333
391
1A
73, 62, 56, 82
74
Bra018250
374
1A
Bol041035
342
1A
95, 62, 50, 73
77
Bra021735
339
11
Bra030637
381
1A
15
Bol008830
102
12
77
Bra005971
160
11
16
Bol009156
188
11
93
76
Bra033464
203
11
At3g51960
228
12
17
Bol009713
383
4A
85, 97
88
Bra016389
368
4A
At1g22070
384
11
Bra031364
378
4A
18
Bol010390
198
11
94, 83, 83
87
Bra019715
193
11
At1g13600
196
11
Bol031441
195
11
78, 98, 98
84
Bra026895
195
11
Bra026896
195
11
19
Bol010836
134
12
98, 81
78
Bra003500
134
12
At3g62420
146
12
Bol044598
141
12
81, 98
88
Bra007679
141
12
Bol033132
171
11
97, 88
82, 56
Bra020735
171
11
At3g30530
173
11
Bol043053
172
12
85, 98
88, 59
Bra025418
172
11
At5g38800
165
12
20
Bol011470
363
3A
95
69
Bra037382
367
3A
At4g01120
360
3A
21
Bol011683
96
12
Bol037733
106
12
22
Bol011719
432
6A
92, 83
78
Bra005287
438
6A
At2g36270
442
6A
Bra017251
396
6A
23
Bol012142
160
6A
61, 95
61
Bra003755
179
6A
At1g75390
173
11
Bol039324
160
6A
Bra008192
165
6A
Bol039895
178
6A
94, 62
76
24
Bol012472
170
5B
97, 85, 84
79
Bra024478
155
5B
At2g18160
171
5B
Bol041488
169
5B
86, 85, 96
83
Bra037235
168
5A
Bra039631
168
5A
25
Bol012703
236
12
95, 72
73
Bra037290
239
12
At2g16770
249
12
Bol042686
244
12
80, 89
71
Bra013048
239
12
26
Bol013712
265
11
87, 63
61
Bra011580
231
12
At4g35040
261
4B
Bol034645
255
11
64, 98
76
Bra034668
255
11
27
Bol012855
294
6A
88
54
Bra033719
266
11
At5g44080
315
5B
28
Bol013623
416
1A
89
75
Bra011485
439
1A
At4g34000
454
1A
Bol033853
410
11
71
55
29
Bol013680
154
11
98, 89, 86
81
Bra011545
179
5B
At4g34590
159
5B
Bol024237
148
5B
89, 97, 78
82
Bra017664
153
5B
Bol034676
142
11
84, 82, 98
78
Bra034639
142
11
30
Bol014051
171
3A
66, 54, 66
68, 64
Bra005335
422
3A
At1g32150
389
3A
Bol022259
422
3A
51, 50, 85
70, 62
Bra023012
403
3A
At2g35530
409
3A
Bol027451
392
3A
96, 66, 64
63, 83
Bra023243
352
3A
Bol039799
400
3A
62, 85, 55
58, 73
31
Bol015239
391
12
87
Bra033649
414
1A
32
Bol016052
394
12
72
Bra010722
445
4B
33
Bol016288
374
11
96, 74
72
Bra027885
373
1A
At1g58110
374
11
Bra035464
176
1B
34
Bol016432
289
11
79, 98
84
Bra009793
291
11
At5g24800
277
5B
Bol022397
287
11
93, 77
81
Bra020471
289
11
35
Bol016607
142
12
94
78
Bra010035
142
12
At5g49450
145
11
Bol032354
139
12
80
76
36
Bol003614
353
1A
80, 50, 70
54, 72
Bra001742
355
1B
At1g49720
403
1A
Bol016788
307
1B
52, 87, 52
61, 48
Bra018800
368
1B
At3g19290
432
1A
Bol018082
133
12
83, 64, 96
59, 91
Bra037533
388
1A
Bol031002
391
1A
74, 57, 88
53, 74
37
Bol017068
187
12
93, 83, 73
60
Bra013005
182
12
At5g60830
206
12
Bol036259
210
12
73, 85, 95
60
Bra029353
104
12
Bra035957
184
12
38
Bol018521
442
1A
75
57
Bra033582
446
11
At4g38900
553
1A
39
Bol018596
243
1B
69, 95
70
Bra011780
246
1B
At4g37730
305
11
Bol028894
246
1B
94, 66
72
Bra017850
240
1B
40
Bol018688
281
11
73, 51, 92
67
Bra010504
222
11
At4g35900
285
5A
Bol029042
270
5B
70, 66, 64
62
Bra011648
262
5A
Bol029939
265
11
90, 59, 66
66
Bra017735
259
5B
41
Bol020032
89
11
78, 76, 100
82
Bra017359
174
11
At2g04038
166
11
Bol032575
176
11
91, 81, 78
69
Bra025144
170
5B
Bol042729
170
5B
80, 97, 78
77
Bra026523
89
11
42
Bol020390
389
11
88
Bra000102
366
11
43
Bol021255
194
4B
77, 73, 97
79
Bra006324
181
4A
At5g15830
186
4A
Bol034371
178
4A
93, 71, 82
75
Bra008670
183
4B
Bol030487
187
4B
70, 93, 75
73
Bra023540
188
4B
44
Bol021964
190
12
64
64
Bra036025
190
12
At3g49760
156
12
Bol037334
186
12
93
64
45
Bol022925
148
5B
97, 84, 88
92
Bra001671
150
5B
At3g17609
149
5B
Bol030865
145
5B
86, 97, 84
88
Bra021258
146
5B
Bol038660
150
11
81, 78, 96
83
Bra022225
116
12
46
Bol023161
624
10
91, 87
59
Bra023224
593
10
At3g10800
675
10
Bra034147
629
10
47
Bol023356
318
5A
96, 84
80
Bra030663
320
5A
At1g06850
337
5A
Bra031541
324
5A
48
Bol024704
162
5B
85, 94
84
Bra008976
164
5A
At5g11260
168
5B
Bol043589
164
5B
90, 88
87
Bra023317
166
5A
49
Bol026864
459
11
97
74
Bra025743
462
11
At1g19490
471
11
50
Bol027526
791
12
97
83
Bra015646
339
12
At1g77920
368
1B
51
Bol027732
371
6A
67
Bra015847
358
6A
52
Bol028631
120
11
97
73
Bra038341
120
12
At1g68880
138
12
53
Bol028975
313
3A
87
82
Bra010572
313
3A
At4g36730
315
3A
96
Bra011701
313
3A
54
Bol033486
303
11
65
55
Bra034925
233
2B
At1g42990
295
10
55
Bol033489
250
11
84, 96
70
Bra032191
330
3A
At1g43700
341
11
Bol043246
330
3A
99, 82
69
Bra034916
263
11
56
Bol033493
310
1B
97
71
Bra034913
222
1B
At1g35490
300
1A
57
Bol037803
266
11
58
Bol040859
266
5A
93
64
Bra015281
268
5A
At1g03970
270
5A
59
Bol041278
333
11
93
80
Bra019436
336
11
At3g44460
12
60
Bol043859
149
12
97
Bra009288
147
12
61
Bol044292
464
10
67
Bra014680
438
10
62
Bol045190
385
4A
87
51
Bra040260
364
11
At1g45249
427
1A
63
Bol045877
386
3A
85
Bra031173
387
3A
Length: amino acid length of bZIP proteins. Identity 1: homology betweenB. oleracea and B. rapa. Identity 2: homology between B. oleracea and A. thaliana.Table 2
Number of bZIP transcription factors in each group based on UARR and LCRs.
Group
Classification domain
bZIP number inB. oleracea
bZIP number inB. rapa (Hwang et al.∗)
bZIP number inA. thaliana (Hwang et al.∗)
Group 1
Q-rich domain
13
16
10
Group 2
D-rich domain
0
4
3
Group 3
P-rich domain
12
12
6
Group 4
N-rich domain
5
9
4
Group 5
S-rich domain
13
18
14
Group 6
G-rich domain
7
6
2
Group 10
Transmembrane domain
4
4
4
Group 11
Several LCRs
38
41
17
Group 12
No LCR or UARR
27
26
13
Total
—
119
136
73
∗See reference [16].


## 3.3. CandidateBolbZIP 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 ofBolbZIP gene in twoB. oleracea inbred lines, cold-tolerantBN106 and cold-susceptibleBN107.BolbZIP genes were selected from an RNA sequencing dataset based on their annotations and their expression profiles were analyzed (data not shown). Among the 119BolbZIP 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 inBN106 and of 7 genes inBN107, whereas 15 genes were downregulated inBN106 and 8 genes were inBN107 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 withinBN106 lines and therefore not calculated (Table 3). Finally, 47BolbZIP 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, 25BolbZIP 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 exceptBol021255. 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 severalBolbZIP genes were comparable between the two lines. However, no significant change in the expression ofBol008071, Bol033132, and Bol042729 was observed in response to cold treatment inBN106, whereas these genes were upregulated at all temperatures inBN107 (Figure 2(a)). By contrast,Bol009713, Bol013712, Bol016432, and Bol022925 were upregulated inBN106, but not inBN107 (Figure 2(b)). The increased expression of17 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 bothBN106 andBN107 (Figure 2(d)). Homologs of cold stress-responseBrbZIP genes were included in the qRT-PCR [16]. These expression patterns are summarized in Figure 4. Moreover, several genes includingBol016432,Bol022925,Bol026864,Bol027732, andBol028975 displayed differential expression between cold (4°C) and freezing (−2°C) temperature. The expression level of the 3 genes,Bol008071,Bol033132, andBol042729, was significantly increased inBN107 under cold conditions and was unchanged inBN106. Among three genes,Bol033132 has 97% sequence similarity toBra020735 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.Table 3
Cold-treatment induced change in expression based on RNA sequencing data. The differentially expressed genes determined based on fold change (FC) ≥2 are displayed with bold font and ≤0.5 with italic font.
Locus_ID
FC1
FC2
Contigslength (bp)
BRADBol number
CDSlength (bp)
A. thaliana homologs
Publishedname
(BN106)
P value
(BN107)
P value
Locus_01882
2.18 ± 0.11
0.0031
1.48 ± 0.02
0.0053
1948
Bol009713
1152
AT1G22070
Locus_01909
0.16 ± 0.01
0.0052
0.65 ± 0.05
0.0105
1555
Bol001886
921
AT2G42380
Locus_04358
3.84 ± 0.02
0.0002
NC
NC
1583
Bol044598
426
AT3G62420
Locus_05013
4.58 ± 0.21
0.0158
1.89 ± 0.51
0.0044
1207
Bol012472
513
AT2G18160
GBF5
Locus_06292
2.35 ± 0.05
0.0044
1.20 ± 0.03
0.0474
1081
Bol013712
798
AT4G35040
Locus_08860
13.09 ± 0.32
0.0006
2.08 ± 0.11
0.0002
1541
Bol027526
2376
AT1G77920
TGA7
Locus_10723
2.99 ± 0.19
0.0012
2.28 ± 0.35
0.0077
1579
Bol026864
1380
AT1G19490
Locus_10986
0.06 ± 0.00
0.0062
0.76 ± 0.04
0.0017
1117
Bol016607
429
AT5G49450
Locus_11058
0.57 ± 0.03
0.0177
0.60 ± 0.04
0.0497
1354
Bol004832
903
AT2G42380
Locus_11330
0.27 ± 0.01
0.0133
1.51 ± 0.33
0.0628
775
Bol042729
513
AT2G04038
Locus_12559
0.35 ± 0.01
0.0090
0.87 ± 0.07
0.0994
1451
Bol028975
942
AT4G36730
GBF1
Locus_14643
0.83 ± 0.19
0.2500
0.32 ± 0.10
0.0346
816
Bol033132
516
AT3G30530
Locus_14780
4.80 ± 0.93
0.0083
0.78 ± 0.03
0.0182
1882
Bol014051
516
AT1G32150
Locus_15053
0.15 ± 0.00
0.0049
0.47 ± 0.03
0.0260
1757
Bol011470
1092
AT4G01120
GBF2
Locus_16059
4.67 ± 2.83
0.0358
1.29 ± 0.28
0.3559
873
Bol027732
1116
Locus_18258
1.46 ± 0.81
0.3124
NC
NC
1013
Bol011719
1299
AT2G36270
ABI5
Locus_19284
0.48 ± 0.02
0.0023
1.00 ± 0.16
0.0535
1580
Bol006077
1179
AT4G02640
BZO2H1
Locus_19975
5.14 ± 0.02
0.0015
3.11 ± 0.24
0.0000
1113
Bol028894
741
AT4G37730
Locus_20038
2.25 ± 0.04
0.0002
0.74 ± 0.09
0.0017
2250
Bol033853
1233
AT4G34000
ABF3
Locus_21455
2.15 ± 0.03
0.0012
1.32 ± 0.05
0.0174
1248
Bol041488
510
AT2G18160
GBF5
Locus_22202
2.90 ± 0.22
0.0078
0.67 ± 0.03
0.0061
1566
Bol000879
936
AT2G46270
GBF3
Locus_22929
0.27 ± 0.05
0.0569
0.58 ± 0.11
0.0236
890
Bol037803
801
Locus_25534
7.11 ± 1.40
0.0024
1.87 ± 0.19
0.0400
1165
Bol039895
537
AT1G75390
Locus_27120
0.13 ± 0.16
0.0645
NC
NC
545
Bol008071
606
Locus_28516
NC
NC
NC
NC
284
Bol033493
933
AT1G35490
Locus_31552
0.29 ± 0.05
0.0628
NC
NC
329
Bol006902
720
AT2G41070
DPBF4
Locus_31870
6.75 ± 3.18
0.0743
0.51 ± 0.17
0.1589
386
Bol037733
321
Locus_35274
0.57 ± 0.02
0.0027
0.19 ± 0.22
0.0014
1107
Bol016432
870
AT5G24800
BZO2H2
Locus_35336
0.12 ± 0.00
0.0113
0.17 ± 0.04
0.0006
969
Bol021255
585
AT5G15830
Locus_35982
4.94 ± 0.07
0.0010
3.61 ± 0.30
0.0016
1216
Bol034676
429
AT4G34590
ATB2/GBF6
Locus_36644
0.40 ± 0.06
0.0362
0.70 ± 0.07
0.1179
1588
Bol008040
1143
AT5G65210
TGA1
Locus_38207
0.56 ± 0.06
0.0396
0.18 ± 0.21
0.0272
673
Bol005115
1032
AT2G40620
Locus_38300
1.23 ± 0.64
0.5000
0.00
NC
318
Bol018596
732
AT4G37730
Locus_38533
4.51 ± 0.34
0.0023
0.85 ± 0.09
0.0454
1978
Bol043707
1095
AT5G10030
TGA4
Locus_38636
9.75 ± 0.87
0.0272
0.56 ± 0.05
0.0346
487
Bol030865
438
AT3G17609
HYH
Locus_39177
1.20 ± 0.18
0.1749
0.38 ± 0.01
0.0054
839
Bol043589
495
AT5G11260
HY5
Locus_39837
0.78 ± 0.05
0.0267
2.20 ± 1.21
0.0097
1648
Bol041035
1038
AT1G06070
Locus_39980
NC
NC
NC
NC
478
Bol038660
453
AT3G17609
HYH
Locus_41080
0.07 ± 0.04
0.0033
2.32 ± 0.80
0.0840
677
Bol010390
597
AT1G13600
Locus_44632
NC
NC
NC
NC
256
Bol029939
798
AT4G35900
FD-1
Locus_44950
2.86 ± 0.00
0.0052
1.32 ± 0.05
0.0301
1447
Bol024526
981
AT5G06960
TGA5/OBF5
Locus_45018
NC
NC
0.70 ± 0.02
0.0223
667
Bol022925
447
AT3G17609
HYH
Locus_46951
0.15 ± 0.17
0.0257
NC
NC
462
Bol020032
270
AT2G04038
Locus_47897
NC
NC
NC
NC
458
Bol037334
561
AT3G49760
Locus_49075
0.39 ± 0.10
0.0149
2.76 ± 0.40
0.0145
739
Bol034371
537
AT5G15830
Locus_55049
NC
NC
0.57 ± 0.29
0.1464
311
Bol018688
846
AT4G35900
FD-1
Locus_56035
0.04 ± 0.00
0.0010
0.15 ± 0.06
0.0055
662
Bol032354
420
AT5G49450
NC, not calculated. FC1, signal intensity of 0°C treated plant over control plant (22°C) inBN106. FC2, signal intensity of 0°C treated plant over control plant in BN107.Figure 2
Relative expression levels of 22BolbZIP genes in cabbage inbred lines cold-tolerantBN106 and cold-susceptibleBN107 under cold stress conditions. 5-week-old plants were treated at 4°C, 0°C, and −2°C. Theactin transcript levels were used for normalization. Values shown are relative to transcript levels in the 22°C treated plants. Error bars indicate standard deviation. (a) Genes showing no significant relative expression change inBN106 and upregulating at all temperatures inBN107. (b) Genes showing upregulation at all temperatures inBN106 and no significant relative expression change inBN107. (c) Genes showing greater upregulation at lower temperatures inBN106 andBN107. (d) Genes showing downregulation in response to cold inBN106 andBN107.Figure 3
RT-PCR analysis of threeBolbZIP genes in response to cold. These genes showed no expression in 22°C-treated cabbage inbred linesBN106 andBN107. Theactin transcript levels were used as an internal control.Figure 4
Heat map representation of cold-responsive expression ofBolbZIP andBrbZIP genes. The expression pattern of the BolbZIPs and their closest BrbZIP homologs in response to cold (4°C) and freezing (−2°C) stresses are shown. Heat map was generated using up- and downregulated gene expression data from qRT-PCR and RT-PCR results.Figure 5
Amino acid sequences of Bol003312 and Bol042749 and their homologs. (a) An alignment of the amino acid sequences of Bol033132 and two homologs, Bra020735 and At3g30530. Conserved sequences of bZIP domain are highlighted using gray shade in the basic and leucine zipper regions. (b) An alignment of the amino acid sequences of Bol042729 and two homologs, Bra025144 and At2g04038.
(a)
(b)


## 4. Discussion

It was known thatB. rapa andB. 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 inB. oleracea and identified 119 BolbZIP proteins and placed them into 63 categories according to sequence similarity (Table 1). To identify the bZIP proteins inB. 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. InArabidopsis, 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 ofB. oleracea,B. rapa, andA. 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 theB. 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 ofB. oleracea. Among them, chromosome 04 was found to contain the highest number ofBolbZIP genes (21%), while chromosomes 05 and 06 harbored the fewest (6-7%) (Figure 1, Table S3). InB. rapa, the highest number ofBrbZIP genes was detected in chromosome 09 (21%) [16]. Additionally, mostBolbZIP genes were distributed in the arm end of each chromosome. The clustered distribution pattern of theBolbZIP 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-responsiveBolbZIP genes, we tested the transcription patterns ofBolbZIP genes enhanced or decreased by cold treatment in twoB. oleracea lines that showed different cold tolerance [16]. Based on their expression patterns, the cold-responsiveBolbZIP transcription factors were divided into four groups (Figure 2). We found that the expression of three genes,Bol008071,Bol033132, andBol042729, was upregulated in cold-susceptibleBN107 but not changed in cold-tolerantBN106. Additionally, when compared with 6 genes published for significant BrbZIP factors involved in the cold response, 4BolbZIP genes (Bol004832, homologous toBra000256, Bra004689, andBra003320;Bol033132, homologous toBra020735;Bol018688, homologous toBra011648; andBol021255, homologous toBra023540) showed similar patterns of expression in response to cold treatment. For example,Bol033132 showed an expression pattern like that of its homologBra020735, indicating that these genes might be conserved key regulator in cold stress responses. Moreover,Bol033132 andBol042729 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 fromB. oleracea provides new opportunities for functional analyses, which may be used in further studies for improving stress tolerance in plants.



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*Source: 4376598-2016-05-23.xml*