Evolution and Expression Divergence of E2 Gene Family...

Research ArticleEvolution and Expression Divergence of E2 Gene Family underMultiple Abiotic and Phytohormones Stresses in Brassica rapa

Nadeem Khan ,1 Chun-mei Hu ,1,2 Waleed Amjad Khan ,1 Emal Naseri,1 Han Ke,1

Dong Huijie,1 and Xilin Hou1

1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Ministry of Science and Technology/College of Horticulture,Nanjing Agricultural University, Nanjing 210095, China2New Rural Research Institute in Lianyungang, Nanjing Agricultural University, 210095, China

Correspondence should be addressed to Chun-mei Hu; [email protected]

Received 2 April 2018; Accepted 25 July 2018; Published 27 August 2018

Academic Editor: Rosaria Scudiero

Copyright © 2018 Nadeem Khan 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.

To understand ubiquitination mechanism, E2s (ubiquitin conjugating enzymes) have crucial part as they play a major role inregulating many biological processes in plants. Meanwhile, Brassica rapa is an important leafy vegetable crop and thereforeits characterization along with the expression pattern of E2s under various stresses is imperative. In this study, a total of 83genes were identified in B. rapa and were classified into four different classes based on domain information. Here, we analyzedphylogenetic relationships, collinear correlation, gene duplication, interacting network, and expression patterns of E2 genes in B.rapa. Furthermore, RT-PCR analysis for 8 multiple abiotic and hormone treatments (namely, ABA, GA, JA, BR, PEG, NaCl, andheat and cold stress) illustrated striking expression pattern under one or more treatments, speculating that these might be stress-responsive genes. The cis-elements and interaction network analyses implicate valuable clues of important function of E2 genesin development and multiple stress responses in B. rapa. This study will further facilitate functional analysis of E2s for improvingstress resistance mechanism in B. rapa.

1. Introduction

Among the eukaryotes, ubiquitination is considered oneof the important types of posttranslational modification ofproteins. It plays crucial role in plant developmental aspectswith holding imperative association, such as regulating anumber of biological process [1], responding to plant bioticand abiotic factors, light regulation, phytohormones, flowersdevelopments, and others several key factors [2–5]. Thecomplex nature of ubiquitination generally is categorizedinto three types of enzymes, namely, ubiquitin-activatingenzyme (E1), ubiquitin conjugating (UBC) enzyme (E2),and ubiquitin-ligase enzyme (E3) as with target protein theubiquitin binds covalently during this process [6]. Except forubiquitin E1, the other two E1 and E2 enzymes are linkedin an ATP-dependent manner and are activated ubiquitin,transfer targeted protein aided by E3 after passing it toactive-site cysteine [6]. The targeted protein can be alsomodified through sequential ubiquitination cycles as the

additional ubiquitin is further ligated into initial ubiquitin,which ultimately forms a polyubiquitin chain, helping themin targeted protein modification [6]. For generating otherbiological activities, then the substrates can be degraded.Due to the attachment of ubiquitin with targeted protein,E2 is considerably important and plays a crucial role inubiquitination [6].

On a global scale the crop productivity is always uncertaindue to climatic changes and series of other biotic-abioticfactors that damage the plant growth and production sub-stantially and lead to severe crop losses. Some of the recentstudies highlighted that involvement of E2 genes familyexited as multifunctional and playing key parts in variousphysiological activities. In several crops, such as in grapes43 [7], tomato 59 [8], rice 39 [9], maize 75 [10], Caricapapaya 34 [11], and Arabidopsis 41 [12], genes were identified,respectively, as during the process of evolution the numberof genes of E2 increases with the complex nature and thedevelopment of organism [13]. For example, fewer genes ≤

HindawiBioMed Research InternationalVolume 2018, Article ID 5206758, 18 pageshttps://doi.org/10.1155/2018/5206758

http://orcid.org/0000-0002-3007-2772

http://orcid.org/0000-0002-5672-3115

http://orcid.org/0000-0002-0945-6165

https://doi.org/10.1155/2018/5206758

2 BioMed Research International

20 in algae were found in ancestral eukaryotes comparedto certain plants and animals > 40 [14]. The increase innumbers and the diversification of E2s are typically governedand have been associated with the eukaryotic evolution [15].The biological diversity drives mainly with the subsequentidentification of novel molecular functions [16]. However,functional analysis of E2s family in higher plants is limitedon the basis of extensive studies. For example, in transgenicArabidopsis, peanut, and soybean the E2 genes respond todrought stress and salt tolerance [17–19], while in mung beanVrUBC1 were found to respond to the osmotic stress [20].Tolerance of UV stress and as well the activation of floralrepressor genes such as AtUBC1 and AtUBC2 is important[21]. The E2 genes family are specifically important fromthe above studies and their further characterization willshed light on their action mechanism. Furthermore, thismay help in the distribution of ubiquitin-proteasome withfundamental understanding.

Chinese cabbage belonging to genus Brassica is an impor-tant leafy vegetable crop being grown worldwide. GenotypeChiifu 401-42 (Chinese cabbage) was recently sequenced andassembled. Since the divergence of B. rapa from Arabidopsis13 to 17 MYA it experienced a whole genome triplication(WGT) event and the data exhibits a close evolutionaryrelationships [22, 23]. During this study, we carried outgenome-wide analysis of BraE2 and its expression divergencepatterns. The conserved motifs, coregulatory network, geneduplications, and in silico transcriptome for various tissueswere conducted. On the other hand, we also investigatedand validated the BraE2 expression patterns under multipleabiotic and hormone treatments. We attempted a completepicture of BraE2 in B. rapa and these results will furtherprovide guidelines for functional analysis of BraE2 eachgenes.

2. Material and Methods

2.1. E2 Ubiquitous Conjugating Enzymes Identification. TheB. rapa sequences were downloaded from the database(http://brassicadb.org/brad/), while for other species rice [9](http://rice.plantbiology.msu.edu/), tomato [8] (https://solge-nomics.net/organism/Solanum lycopersicum/genome), Maize[10] (https://www.maizegdb.org/), Vitis vinifera [7], and Car-ica papaya [11] sequences were downloaded based on thereported studies. From Pfam database (https://pfam.sanger.ac.uk/) the E2 UBC domains (PF00179) were first down-loaded and Hidden Markov Model (HMM) search wascarried out in Brassica genome with default parameters.After identifications, we verified these potential sequencesusing NCBI database (https://www.ncbi.nlm.nih.gov/) andSMART (http://smart.embl-heidelberg.de/). The theoreticalindex (pI), GRAVY (Grand Average of hydropathicity),protein length (aa, and molecular weight were examinedthrough ExPASy protparam (http://web.expasy.org/protpar-am/) and, for subcellular predication, we explored WoLFPSORT server (https://wolfpsort.hgc.jp/).

2.2. Phylogenetic Analysis and Gene Duplications. To studythe evolutionary relationship of BraE2, we performed

multiple sequence alignment using CLUSTALW with defaultparameters in MEGA 7.0. A phylogenetic tree was con-structed using Maximum Likelihood Method (MLM) withJones-Taylor and Thornton amino acid substitution method(JTT) using MEGA 7.0 [24] and was performed with 1000bootstrap replication. We also examined the Ks/Ka (syn-onymous and nonsynonymous) values among the duplicatedgenes with the help of MEGA 7.0. The coding sequences ofduplicated pairs of BraE2 were first aligned by following theNei and Gojobori model performed in MEGA 7.0 [24].

2.3. Motif and Gene Structure Analysis. The conserved motifwas analyzed through MEME (http://meme-suite.org/) alimit of 20 motifs with minimum width ranges from 10and maximum 120, and the others parameter were set asdefault. For genes structure analysis, we explored Gene Struc-ture Display Server (http://gsds.cbi.pku.edu.cn/) (GSDS) andthe exon-intron organization of BraE2 was determinedby comparing the coding and its corresponding genomicsequences.

2.4. Chromosomal Localization and Gene Syntenic andPromoter Analysis. All the information for each gene ofBraE2 was collected from the B. rapa database (http://bras-sicadb.org/brad/) and the images were drawn throughMapchart [25]. Syntenic genes were also identified amongthe three subgenomes (LF,MF1, andMF2) ofB. rapa database.All the identified BraE2 ubiquitin enzymes, 15 kb promotersequence, were analyzed through PlantCARE database(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)for the identification of cis-regulatory elements [26].

2.5. Expression Pattern Analysis and Interacting Networkof BraE2 Genes. For expression profiling of BraE2 amongvarious five tissues (root, stem, leaf, flower, and silique), weanalyzed B. rapa accession (Chiifu-401-42). Based on thepreviously reported RNA-seq data [27] for gene expressionpatterns were utilized and the fragments per kilobase of exonmodel per million mapped (FPKM) values were transformedinto log2. Finally, heat maps were generated for all theBraE2 and for their paralogous pairs using omicshare tools(http://www.omicshare.com/). For interaction network, weexplored string (https://string-db.org/).

2.6. RNA Isolation and qRT-PCR Analysis. We isolated theRNA from the treated leaves of B. rapa with the help ofRNAkit (RNAsimply total RNAkit; Tiangen, Beijing, China)according to the manufacturer’s instructions. After that, forevery RNA sample, we checked the quality and quantityby using an agrose gel. With the help of Prime Script RTreagent kit (TaKaRa) the RNAwas then reversely transcribedinto cDNA. For qRT-PCR analysis, gene specific primerswere designed by Beacon Designer 8.0 and are listed inSupplementaryTable 1). The reactions were performed usingStep one plus Real-Time PCR System (Applied Biosystems,Carlsbad, CA), with the help of the following parameters:94∘C for 30 s, 40 cycles at 94∘C for 05 s, and 60∘C for 15s and 72∘C for 10 s. In order to check the specificity of theamplification melting curve was generated with following

http://brassicadb.org/brad/

http://rice.plantbiology.msu.edu/

https://solgenomics.net/organism/Solanum_lycopersicum/genome

https://solgenomics.net/organism/Solanum_lycopersicum/genome

https://pfam.sanger.ac.uk/

https://pfam.sanger.ac.uk/

https://www.ncbi.nlm.nih.gov/

http://smart.embl-heidelberg.de/

http://web.expasy.org/protparam/

http://web.expasy.org/protparam/

https://wolfpsort.hgc.jp/



http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

BioMed Research International 3

Class 1 Class 2 Class 3 Class 4

4

5

6

7

8

9

10

Rang

e

pI

Class 1Class 2

Class 3Class 4

(a)

Rang

e

Class 1 Class 2 Class 3 Class 4−1.0

−0.8

−0.6

−0.4

−0.2

0.0

0.2

GRAVY

Class 1Class 2

Class 3Class 4

(b)

Figure 1: (a) Indicating the pI values among different subclasses of BraE2. (b) Showing the grand average of hydropathicity (GRAVY) amongdifferent classes of BraE2.

parameters: 61 cycles at 65∘C for 10 s. The relative geneexpression values were calculated using the comparative Ctvalue method [28].

2.7. Plant Materials and Multiple 8 Treatments. For thisexperiment, we used a typical Chinese cabbage cultivarChiifu 401-42. It has been mostly used for research studies,mainly due to its complete whole genome sequencing. Ingreenhouse of Nanjing Agricultural University, we cultivatedChinese cabbage in potting soil with following controlledenvironmental conditions as follows: 65-70% humidity, light16 h/25∘C, and dark 8 h/20∘C. After a one-month interval,when the seedlings reached five-leaf stage theywere subjectedto multiple abiotic and hormone stresses under continuoustime intervals (1, 6, and 12 hrs). Meanwhile, for multiplehormone treatments plant was treated with four differenttypes such as ABA (100 𝜇M), GA (100 𝜇M), JA (50 𝜇M), andBR (50 𝜇M). Salt stress plants were subjected to 250mM,while drought stress pots were irrigated with 15% (w/v)polyethylene glycol under normal growth conditions. Forsalt and osmotic treatments, irrigation solution was keptstanding for 30mins, respectively. Cold and heat stress plantswere exposed to 4 or 38∘C, while other growth parameterswere set as discussed above. All treatments were based onthree biological replicates at the end leaf samples which werefrozen immediately in liquid nitrogen and stored at −70∘C forfurther analysis.

2.8. Pearson Corelation. The PCCs values for transcriptomicdata and qRT-PCR were performed in excel sheet 2013 basedon the paralogous pairs [29].

3. Results

3.1. Identification and Bioinformatics Analysis of UBC Genesin B. rapa. In the present study, we explored multiple bioin-formatics resources for the identification of UBC (Ubiquitin

Conjugating) genes in B. rapa. A total of 83 genes wereidentified through genome-wide analysis by using HMMandBLAST search methods. All these candidate genes were des-ignated as BraE2-1-BraE2-83 according to generic order andwere classified into fourmajor classes based on domain infor-mation.The characteristic analysis of these 83 genes includingGene ID, cDNA (bp), Chr: Start-End point, Exon, Subcellularpredication, pI, GRAVY (Grand Average of hydropathicity),Protein length (aa), and MW (kDa) were analyzed andare listed in (Supplementary, Table.2) for each protein. Thenumber of amino acid were markedly varied from 111 to 1662in length, with the corresponding molecular weight (MWs)ranging from 12.38 to 185.26 in kDa, respectively, and thetheoretical isoelectric points varied from 4.19 (BraE2-75) to9.83 (BraE2-40) which were further shown in Figure 1(a) fordifferent classes. According to the stability index measuremost of the proteins were unstable with hydrophilic in nature;however two proteins (BraE2-13-BraE2-4) from Class 1 werehydrophobic, suggesting their stability (Figure 1(b)).Majorityof genes were located in different specific-organelles suchas mitochondria, nucleus, cytosol, endoplasmic reticulum,plasma membrane, and other secretory pathways, whereasthey were regulated in variable microenvironment.

3.2. Phylogenetic Analysis and Structure of BraE2 Genes.To investigate the evolutionary relationship and functionaldivergence between BraE2 proteins and known BraE2s fromother species, a maximum likelihood phylogenetic tree wasconstructed on the basis of the full amino acid of BraE2 familyprotein from B. rapa, A. thaliana, and rice (Figure 2(a)).The BraE2 genes represent similarities on the basis of phy-logenetic analysis and contain UBCc (Ubiquitin ConjugatingEnzyme, E2 catalytic) superfamily domain. Furthermore, onthe basis of domain information BraE2 family were furtherdistributed into four major classes such as Class 1 containonly UBC catalytic domain, Class II (N-terminal extension),Class III (C-terminal extension), and Class IV contains


BraE

2-45

BraE

2-52

45

BraE

2-43

31

BraE

2-46

BraE

2-48

AT1G

5302

548

67

99

BraE

2-44

BraE

2-47

BraE

2-42

AT3G

1535

5

3850

86

97

OsU

BC42

OsU

BC43

93

38

OsUB

C41

OsUB

C39

OsUBC

40

4556

55

OsUBC

37

33

OsUBC

36Br

aE2-

50AT2G

3377

0

98

OsUBC35

87

OsUBC34

BraE2-5

1

AT2G16920

BraE2-49

BraE2-53

4573

60614951

99

AT3G12400

AT5G13860

99

41

AT3G17000

OsUBC45

99 OsUBC46

AT1G17280

AT5G50430

819991

18

AT5G42990

AT1G45050

31BraE2-55

25 BraE2-54

AT1G75440

8617

OsUBC2616

BraE2-59

OsUBC25

4718

BraE2-57

BraE2-56AT4G36410

6696

49

BraE2-5899

AT3G52560AT2G36060

71

AT1G70660AT1G2326055

99

BraE2-24AT3G55380

40

BraE2-25

43

BraE2-64

60

OsUBC11OsUBC12

62

BraE2-65BraE2-66AT3G46460AT5G59300

4436

73

53

98

44

7

5

BraE2-38AT3G20060

36

BraE2-39

55

AT1G50490

23

OsUBC27

21

BraE2-35BraE2-36

32

42

BraE2-69

99

BraE2-61BraE2-62

52

BraE2-60

37AT3G57870

81

OsUBC1

OsUBC2

6456

BraE2-63

56

OsUBC3

99

BraE2-83

BraE2-30

AT5G62540

93BraE2-41

OsU

BC7

OsU

BC964

OsU

BC853

BraE2-3432

BraE2-27

BraE2-26

AT1G14400

31BraE2-29

76

BraE2-28BraE2-32BraE2-33

0672

0G2T

A29

5666

5029

2536

4997

276

19

BraE

2-72

BraE

2-73

76

OsU

BC33

99

BraE

2-70

BraE

2-71

89

AT5G

2576

0

OsU

BC32

4499

36

19

BraE

2-19

BraE

2-68

64

BraE

2-21

53

BraE

2-22

25

BraE

2-20

49

AT1G

7887

0

37

OsUBC

47

71

AT1G

1689

0

99

OsUBC

44

BraE

2-16

AT5G50

870

9599

20

BraE2-7

4

BraE2-7

5

87

AT5G41

340

33

AT1G63800OsU

BC10

41 92

BraE2-67

BraE2-76AT2G46030

43 43 99

BraE2-23AT3G24515

96

OsUBC4899

OsUBC5OsUBC6 43OsUBC4BraE2-80AT2G18600 47BraE2-77AT4G36800 77BraE2-82 53BraE2-81BraE2-78

BraE2-79 64 7192

8574

9918

13

7

63

BraE2-15BraE2-40

97

AT1G3634099

BraE2-18AT3G13550

99

BraE2-37AT2G32790 99

336

86

AT3G08700

OsUBC18 5

11

BraE2-6

BraE2-12

86

BraE2-9

AT5G5615027

40

BraE2-31

30

OsUBC16

9

BraE2-2

BraE2-3

65

BraE2-1

93

AT3G08690

78

OsUBC22

OsUBC23

97

24

OsUBC17

8

BraE2-11

BraE2-14

77

AT2G16740

87

OsUBC13

50

OsUBC14

13

BraE2-17

AT1G6423046

BraE2-10

47

OsUBC15

8

AT5G41700BraE2-13

AT4G2796059

BraE2-471

BraE2-5AT5G53300

32BraE2-7

BraE2-873

4330

714

15

3

0

Brassica rapaArabidopsis thalianaRice

(a)

16.87%

13.25%4.82%

65.06%

Class-1Class-2

Class-3Class-4

(b)

9.09%

11.5%

15.78%20.05%

10.43%

10.96%22.19%

Arabidopsis thalianaBrassica rapa

RiceMaize

TomatoVitis ViniferaCarica Papaya

(c)

Figure 2: Continued.


BraE

2-46

BraE

2-48

56

AT1G

5302

5

63

BraE

2-43

43

BraE

2-45

BraE

2-52

30

79

BraE

2-44

BraE

2-47

BraE

2-42

AT3G

1535

5

46599492

BraE

2-50

AT2G

3377

0

99

BraE

2-51

BraE

2-53

BraE2-4

9AT2G

1692

0

3850

36

49

98

AT3G12

400

AT5G13860

100

10

AT3G52560

AT2G36060

95AT1G23260

39

AT1G70660

100

BraE2-24

AT3G55380

54

BraE2-2555

BraE2-6441

BraE2-65

BraE2-66

AT3G46460

AT5G5930038

45

90

99

25

5

AT1G17280

AT5G5043099

AT3G17000

94

BraE2-58

BraE2-59

AT1G45050

35

AT5G42990

19

BraE2-55

20

BraE2-54

AT1G75440

57

BraE2-56

BraE2-57AT4G36410

95

99

43

80

99

20

9

BraE2-38AT3G20060

25

AT1G50490

23

BraE2-39

40

BraE2-35BraE2-36

25

20

BraE2-69

99

BraE2-61BraE2-62

65

BraE2-60

72

AT3G57870

89BraE2-63

100BraE2-83

BraE2-30

AT5G62540

83

BraE2-3435

BraE2-41

BraE2-27

BraE2-26

BraE2-2930

AT1G14400

73

BraE2-28

BraE2-32

BraE2-33

AT2G02760

27

54

71

60

42

25

46

95

40

35

27

27-2Ear BBraE

2-73

100

AT5G

2576

0

BraE

2-70

BraE

2-71

57 100 20

10

BraE

2-16

AT5G

5087

0

100

AT1G

1689

0

BraE

2-20

BraE

2-22

AT1G

7887

0

22

BraE

2-21

BraE

2-19

BraE

2-68

61 55

16

20 97 31

4

BraE2-1

5

BraE2-4

0

95

AT1G36

340

95

BraE2-1

8

AT3G13550100

BraE2-37AT2G32790

98

36BraE2-6

BraE2-12 77BraE2-9AT5G5615027

27

BraE2-3137

AT3G0870015

BraE2-2

BraE2-367

BraE2-188

AT3G08690

67

BraE2-11

BraE2-14

74

AT2G16740

89

AT5G41700

26

BraE2-17

AT1G64230

53

BraE2-10

63

BraE2-13

AT4G27960

50

BraE2-4

67

AT5G53300

BraE2-5

BraE2-7

BraE2-8

72

31

35

24

10

38

21

91

44

334

BraE2-74

BraE2-75

86

AT1G63800

51

AT5G41340

87

BraE2-67

BraE2-76

AT2G46030

5060

99

BraE2-23AT3G24515

99BraE2-80

AT2G1860052

BraE2-77AT4G36800

30BraE2-82

22BraE2-81

BraE2-78BraE2-79

63

63

59

100

21

14

Brassica rapaArabidopsis thaliana

Br06

Br07

Br05Br04

Br03

Br02

Br01

Br09

Br10

At01 At02

At03

At04

At05

Br08

(d)

Motif 1-20

BraE2-1BraE2-10BraE2-11BraE2-12BraE2-13BraE2-14BraE2-15BraE2-16BraE2-17BraE2-18BraE2-19BraE2-2BraE2-20BraE2-21BraE2-22BraE2-23BraE2-24BraE2-25BraE2-26BraE2-27BraE2-28BraE2-29BraE2-30BraE2-31BraE2-32BraE2-33BraE2-34BraE2-35BraE2-36BraE2-37BraE2-38BraE2-39BraE2-4BraE2-40BraE2-41BraE2-42BraE2-44BraE2-46BraE2-47BraE2-48BraE2-49BraE2-5BraE2-50BraE2-51BraE2-52BraE2-53BraE2-54BraE2-55BraE2-56BraE2-57BraE2-58BraE2-59BraE2-6BraE2-60BraE2-61BraE2-62BraE2-63BraE2-64BraE2-65BraE2-66BraE2-67BraE2-68BraE2-69BraE2-7BraE2-70BraE2-71BraE2-72BraE2-73BraE2-74BraE2-75BraE2-76BraE2-77BraE2-78BraE2-79BraE2-8BraE2-80BraE2-81BraE2-82BraE2-83BraE2-9

0kb 1kb 2kb 3kb 4kb 5kb 6kb 7kb 8kb 9kbCDS upstream/ downstream Intron

5

3

(e)

Figure 2: (a) Phylogenetic relationships of BraE2 among three species B.rapa, A.thaliana, and rice.The phylogenetic tree was constructed byMEGA 7 using the Maximum Likelihood Method (1000 bootstrap). Genes of different species are marked with different colors. (b) Relativeclassification patterns of BraE2 family based on the number of genes among different classes. (c) Showing classification pattern of E2s genes indifferent species. (d) Phylogenetic tree and Circos plotter family. (A)The phylogenetic tree was constructed byMEGA 7 using the MaximumLikelihood Method (1000 bootstrap). (B) The Circos plotter family between B. rapa and A. thaliana were elucidated by MCScanX program.(e) The conserved motif was constructed by MEME program. The intron, upstream/downstream, and CDS region are represented by pink,brown and line and green boxes, respectively.The bottom of the figure the relative position is proportionally displayed based on the kilobasescale.


(both N- and C-terminal extensions) [30, 31]. We foundthat Class I represents the highest 54 members, followedby Class IV, Class III, and Class II with 14, 11, and 4members, respectively, and among all the classes highest 65.06% proportion belonged to Class I (Figure 2(b)). We alsocalculated the proportion of genes in various species basedon previous reported studies (Figure 2(c)). Notably, the B.rapa were found to have highest (22.19%) number of genescompared to other species such as maize (20.05%), tomato(15.78%), Vitis Vinifera (11.5%), Arabidopsis (10.96%), rice(10.43%), and Carica papaya (9.09%). On the other hand, wealso a constructed a phylogenetic tree between B. rapa and A.thaliana with family circle plotter using MCScanX program(Figure 2(d)).

To better understand the structural diversity of BraE2in B. rapa, we compared the intron/exon structure andconserved motifs (Figure 2(e)). The gene structures wereobtained by comparing the genomics and ORF sequences.Moreover, each structure possessed a minimum of 1 and21 maximum intron. Intriguingly, for Class IV the BraE2-49 genes were found to have highest 21 numbers of introncompared to other classes. For majority of genes the commonpattern with 32.5% shared 3-4 numbers of intron/exon. Inaddition, for different classes of BraE2 the intron/exon wasmostly similar in pattern for same classes and for thosewhichwere closely relatedmembers.Therewas only one gene,BraE2-37 with one intron. Furthermore, twenty conservedmotifs were captured using MEME software. Surprisingly,most numbers of motifs 13 were found for Class IV (BraE2-49) gene. Motifs 1, 2, 3, and 4 were present in almost allthe members of BraE2, while other motifs were detected inless than half of BraE2. The regulation patterns for most ofthe complex structure were tight among few members of theBraE2.

3.3. Collinearity Correlation of BraE2 among A. thaliana andB. rapa and Copy Number of Variation. We explored twothings, collinear correlation between B. rapa and A. thalianagenes which were presented in (Figure 3(a)) and the copynumber of variation during B. rapa specific WGT event.Interestingly, the highest number 14 for one-copy variationwas found in Class I, followed by two- and 3-copy variationwith 7 each, respectively, as shown in Figure 3(b) and Sup-plementary Table 3. The B. rapa genome basically containedthree genomes, namely, LF (least fractioned genome), MF1(medium fractioned), and MF2 (more fractioned). Based onthese three genomes, we calculated the number of genes fordifferent classes, although the highest number of genes 19 wasfound in MF1, followed by 18 in LFand 16 in MF2for ClassI compared to other classes (Figure 3(c)). Overall, resultsshowed that different three subgenomes were more similarwith slight variations (Figure 3(d)) and ratio for MF1 washighest 36.25%, followed by LF 32.5% and MF2 31.25%.

3.4. Chromosomal Localization, Gene Duplication and Selec-tive Pressure Analysis of BraE2. The BraE2 chromosomeswere distributed on all the ten (A01-A10) B. rapa genomesexcept from three genes (BraE2-3, BraE2-43 and BraE2-45)which was on scaffold (Figure 4(a)). The distribution pattern

of BraE2 were highly varied, majority of genes 13 were foundon A03, followed by 10 on A09, whereas chromosomes A02and A05 contains 9 number of genes each respectively. Theoverall ratio of these chromosomes (A01-A10) for BraE2 arefurther presented in (Figure 4(b)).

To explore the functional diversification of protein andduplication analysis are significantly important in gene family[32]. In this study, we analyzed mainly two types of dupli-cation (Segmental and tandem) to examine the contributionof duplication events in this family (Supplementary, Table.4). Based on the paralogous pairs of B.rapa genomes, wecalculated Ka/Ks ratio for ten pairs of segmental and oneof tandem array. The results showed that all the pairs hada less than 1.00 values, which indicated that these pairswere purifying in nature. The rate of divergence for all theduplicated pairs with an average of 7.37 (MYA), suggestingthat their divergence occurred during Brassica triplication(5∼9 MYA) event. Furthermore, with the help of MCScanXprogram, we identified the types of duplication.

3.5. Gene Expression Pattern in Various Tissue and SyntenicParalog Pairs Patterns in B. rapa. Since no ubiquitous conju-gating enzymes have been documented in B. rapa previously,we analyzed the expression patterns of BraE2 under 5 variousorgans (roots, stems, leaves, flowers, and siliques) of B.rapa (Figure 5(a). Supplementary Tables 5 and 6). A heatmap for expression patterns was generated by displayingthe expression profiles in clustered for BraE2. Majority ofthe genes showed a striking expression pattern for differentclasses, whereas a few of them exhibited a similar expressionpattern. Moreover, some of the genes, about 11.08%, showedno expression pattern in any tissue, and the rest of themshowed a significant variation in expression pattern in one ormore tissues. The tissue clustered expression pattern was alsoexhibited (Figure 5(b)), and two genes from stem and siliqueseach, respectively, were expressed, suggesting that these genesmay play a specific role in the relevant tissues.

We also investigated trends of expression pattern for25 paralogous pairs (Figure 5(c)). These paralogous pairsshowed a high alteration in expression level in five tissues.Most of these genes showed a high expression patterns,speculating that these paralogous pairs might have similarfunction. Additionally, the PCC values for paralogous pairsacross five tissues were also calculated and a total of 11 pairsshowed a (> 0.6) PCC, suggesting a positive correlation. Ingeneral, we can infer a positive close correlation between twofactors with (> 0.6) PCC. Such positive correlation amongBraE2 pairs probably implicates functional conservation orsubfunctionalization after duplication. There were two pairssuch as BraE2-20-BraE2-21 and BraE2-80-BraE2-83 withnegative PCC and two pairs with no PCC (BraE2-81 BraE2-82, BraE2-58 BraE2-59), implicating that these pairs due topseudogenization might loss function.

3.6. Coregulatory Networks and Expression Profiling of BraE2Genes in Response to Multiple Abiotic/Phytohormones Treat-ments. To analyze the cis-regulatory elements, we utilizedonline tool PlantCARE for the identification of BraE2 genesin B. rapa. Based on our results, we make five categories such


At01

0 5 10 15

20

25

30

At02

0

5

10

15

At03

0

5

10

15

20

At04

0

5

10

15

At05

0

5

10

15

20

25

Br01

0

5

10

15

20

25

Br02 0

5

10

15

2025

Br03

051015202530

Br04

0

510

15

Br05

0

5

10

15

20

25

Br06

0

5

10

15

20

25

Br07

0

5

10

15

20

25

Br08

0

5

10

15

20

Br09

0

5

10

15

20

25

30

35

Br10

0

5

10

15

AT5G41700

AT5G25760

AT5G13860

AT2G33770

AT5G42900AT5G41340

AT2G16

740

AT2G32

790

AT2G02760

AT3G46460

AT1G

5049

0

AT1G

7887

0

AT1G

6380

0

AT3G17000

AT1G

3634

0

AT1G

1440

0

AT5G62540

AT3G52560

AT5G50870

AT1G

1689

0

AT5G50430

AT3G08690

AT4G36410

AT3G12400

AT2G16920

AT5G53300

AT2G46030

AT3G57870

AT1G

7544

0

AT2G36060

AT2G

1860

0

AT1G

4505

0

AT5G56150

AT3G20060

AT1G

2326

0

AT1G

1728

0

AT1G

5302

5

AT4G36800

AT3G08700

AT1G

7066

0

AT4G27960

AT1G

6423

0

AT5G59300

BraE2-42BraE2-39

BraE2-30

BraE2-58

BraE2-66BraE2-9

BraE

2-11

BraE

2-12

BraE2-2

BraE2-23

BraE2-76Br

aE2-

16

BraE

2-79

BraE

2-60

BraE

2-25

BraE2-1

BraE2-38

BraE2-3

6

BraE2-15

BraE2-18

BraE2-5

2

BraE2-46

BraE2-54

BraE2-14

BraE2-24

BraE2-41

BraE2-4

BraE2-29

BraE2-70

BraE2-64

BraE2-26BraE2-80

BraE2-83

BraE2-72

BraE2-5

BraE2-6

BraE2-82

BraE2-56BraE2-13

BraE2-32BraE2-31

BraE2-8

BraE2-78

BraE2-47

BraE

2-7

BraE

2-73

BraE

2-51

BraE

2-81

BraE

2-37

BraE

2-75Br

aE2-

63

BraE2

-50

BraE

2-67

BraE2-44

BraE2-4

0

BraE2-5

9

BraE2-27BraE2-22


BraE2-34

BraE2-49

BraE2-74BraE2-61

BraE2-68

BraE2-77

BraE2-69

BraE2-48

BraE2-21

BraE2-10

BraE2-28

BraE2-26

BraE2-55

BraE2-17

BraE2-20

BraE2-65

BraE2-19

BraE

2-57

AT3G15355AT3G13550

AT3G55380

AT3G24515

SegmentalTandemDispersed

BraE

2-53

(a)

02468

10121416

0 1 2 3 4

Num

ber o

f Gen

es

Copy Variation Class 1 Class 2 Class 3 Class 4

(b)

02468

101214161820

Class 1 Class 2 Class 3 Class 4

Num

ber o

f Gen

es

Subgenomes LF MF1 MF2

(c)



31.25%36.25%

32.5%

LFMF1MF2

(d)

Figure 3: (a) The collinear correlation for all the genes of BraE2 was displayed between B. rapa and A. thaliana. The ten Chinese cabbagechromosomes (Br01-Br10) and the five A. thaliana chromosomes (At1-At5) are shown in different random colors. The illustration was drawnusing Circos Software. For duplication types, tandem array was displayed with green color and dispersed with purple. (b) Showing copynumber of variation among different subclasses of BraE2s. (c) Showing number of genes based on 4 different subclasses of BraE2s. (d) Showingthe ratio of BraE2 genes among three subgenomes of B. rapa.

as Light,Hormones,Heat andCold, andCircadianwhile eachcategory contains related responsive elements (Figure 6(a)).In addition, we calculated the number of genes and findtheir percentage among five categories. A high number ofgenes (39.9%) were found in light category, common lightresponsive elementswith 40 common cis-regulatory elementswere identified (ACE, GAP, LAMP, GTI, GATA, G-Box, ATI,and others), further summarized in Supplementary Table 7.Most of the common regulatory elements were dominated byanother category and about 34.89% genes were involved inboth biotic and abiotic stresses and common cis-regulatorytypes were ARE, Wun-Motif, MBS, TC-Rich repeats, andothers. For hormones category, 17.92% genes participated and13 types such as ABRE, CGTCA, TCA, and GAREmotif werefound in the promoter regions of BraE2, suggesting that itcould affect the expression levels of BraE2 genes in B. rapa. Asubstantial number of light responsive, hormones, heat andcold stress, and other important elements were observed inthe promoter sequences of BraE2. This clearly indicated theirprobable role in both biotic-abiotic factors and hormonalpathways.

The biological and signaling transduction pathway aretypically governed by genes through interaction network.To understand gene family function, the investigation ofpotential network is considerably important [33]. To fur-ther elucidate the interaction network of BraE2 family,proteins-protein interaction were constructed with the helpof STRING software. Most of the genes showed a close andcondense relationship with each other except from a fewgenes as shown in (Figure 6(b)). Among all the BraE2 genesshowed a very dense correlation, suggesting that these genesare involved in several fundamental mechanisms and arefurther regulated by many down/upstream genes.

To study genes function its expression profile provides auseful information with valuable clues for understanding it

[34]. Recent studies have suggested that BraE2 genes havebeen implicated in plant responses to signaling and abioticstresses [9, 19, 35], and the presence of stress-responsive cis-elements in the promoter of the BraE2 genes speculated thattheir involvement in B. rapa is a response to different stresses.To confirm this speculation, the transcriptional expressionsof the 15 pairs of BraE2 were analyzed in B. rapa by qPCRafter application of multiple abiotic and hormone stressessuch as, ABA, GA, JA, BR, PEG, NaCl, and heat and coldstress. Heat map was generated in response to eight multipletreatments for transcript expression fold change as shown inFigures 6(c) and 6(d) and Supplementary Table 8. Most ofthe putative genes were highly expressed and showed highstriking expression patterns. Majority of the genes (55.55%)were upregulated and (44.44%) downregulated in responseto heat treatment, followed by GA (52.22%) upregulation and(47.77%) downregulation. Interestingly, both PEG and NaClshowed similar expression patterns as 50% were up- anddownregulated. Noticeably, BR were found to be sensitive,as most of the genes (74.44%) were downregulated, in caseABA and cold treatment minor changes were exhibitedin the expression patterns as 42.22% and 46.67% wereupregulated (Figure 6(e)). On the other hand, based onthe results of relative expression values, we analyzed thecorrelation and regulatory network among the selected 15pairs of BraE2. For correlation, we categorized the PCCvalues into three sections such as High (>0.6), Medium(>0 and < 0.5), and Negative (< 0). For cold stress, 9PCC values were higher among all, followed by JA, BR,and NaCl with 8 PCCs each value observed, respectively.These results indicate and signify their close relationshipamong each other, whereas for ABA treatment most of the9 PCC values were negative which suggested its contrastingnature to other treatments (Supplementary Table 9 andFigure 6(f)).


BraE2-13BraE2-82

BraE2-56

BraE2-39

BraE2-42

A01

BraE2-9BraE2-30 BraE2-32BraE2-19

BraE2-78


BraE2-58

A02

BraE2-16

BraE2-23BraE2-2BraE2-11BraE2-57

BraE2-37

BraE2-76BraE2-51


BraE2-7 BraE2-81BraE2-73

A03


BraE2-63

BraE2-60

BraE2-79

A04

BraE2-18BraE2-59BraE2-36BraE2-1BraE2-38

BraE2-40

BraE2-44BraE2-15

BraE2-52

A05

BraE2-22BraE2-27

BraE2-71BraE2-35

BraE2-46

BraE2-33

A06

BraE2-34BraE2-74


BraE2-61

BraE2-54BraE2-49

A07

BraE2-29BraE2-41

BraE2-48BraE2-69

BraE2-4BraE2-77 BraE2-21

A08

BraE2-10


BraE2-20

BraE2-62

BraE2-80BraE2-26

BraE2-70BraE2-64

A09

BraE2-65


BraE2-6

A10


Scaffold

(a)



6.25%

12.5%

8.75%10%7.5%

11.25%

8.75%

17.5%11.25%

6.25%

A01A02A03A04A05

A06A07A08A09A10

Chromosomes Distribution

(b)

Figure 4: (a) Chromosome location of the BraE2 was obtained from the GFF file and displayed by using Mapchart. (b)The relative shares ofdifferent subclasses of BraE2 from A01 to A10 chromosomes of B. rapa.

4. Discussion

4.1. Identification, Phylogeny, and Gene Duplication of BraE2Family. In many aspects of plant growth and development,ubiquitin conjugating enzymes are considerably importantand crucial to variety of plant stress responses. In the presentstudy, a total of 83 BraE2 genes were identified with UBCdomain in B. rapa genomes. Based on the comparison withother species, this number was higher as shown by previousreported studies such as 75 identified in maize [10], 59 intomato [8], 50 in human [36], 41 in Arabidopsis [12], and 39in rice [9].The variation in the number of BraE2 genes showsspeculation that during the course of evolution BraE2 familyhad underwent functional divergence. The BraE2 were fur-ther subdivided into four different classes, namely, Class I-IVbased on the domain information with respect to N- and/orC-terminal extensions [30, 31]. Majority of BraE2 genes wereretained, particularly in Class I as there were 14 one-copyvariants and 7 each for two or three copy variations comparedto others classes of BraE2 in B. rapa. As a result a highnumber of BraE2 members were retained in B. rapa genome,after whole genome duplication event (WGD). Expansionof gene family and the adaptation of gene function arereliable on gene duplication as per environmental conditions.Variations in either structural features of coding sequencesor amino acid leads to the functional diversification [37].To understand duplication events, here we analyzed theexpansion mechanism of BraE2 family. The results showedthat 10 pairs from segmental duplication and one pair oftandem duplication were identified based on paralogouspairs, suggesting that segmental duplication contributed tothe expansion of BraE2 family. Our results are further inagreement with the gene dosage hypothesis [38]. In addition,

we also calculated Ka/Ks ratio of these 11 pairs. All thepairs of BraE2 genes indicated less than 1.00 value, stronglyimplicating its purifying selection in nature. Furthermore, theBraE2 diverged 7.37 MYA during the specific Brassica WGTevent. These results suggested that, to overcome the selectivepressure for their survival needs the BraE2 duplicate early,which signify their diverse functions in nature. In the presentstudy, a phylogenetic tree was constructed among threespecies such as B. rapa, A. thaliana, and rice. Our resultedclassification for BraE2 family was consistent with domaininformation. To gain insight into the structural diversity ofthe BraE2 family, gene structure and conserved motifs werealso analyzed. It is well understood that the diversity amongspecies mainly happens through genes organization in thegenome [37]. The structure and function of molecules in asystem can be understand through patterns of motif in thenucleotide or protein sequences [39]. The patterns for mostof the genes were similar in nature and were dominated by 3-4 numbers of intron. The regulation patterns for conservedmotif were tight and specifically motif 1, 2, 3, and 4 werecommon in theBraE2 family. In addition, the process of tissueexpression divergence is mainly associated with expansion offamily and the duplication types in the neofunctionalizationor subfunctionalization models [40–42].

4.2. Expression Divergence, Multiple Abiotic/Hormone Treat-ments, and Interaction Network. In this study, we also exam-ined the tissues-specific expression patterns of BraE2, andmajority of genes showed a high striking expression patternsin all the five tissues or at least several. Some of the geneswere expressed in similar patterns, suggesting their commonimportance in function of plants. In addition, there werefew genes that showed tissue-specific patterns, indicating that


Silique

Root

Flower

Stem

Leaf

BraE2−1BraE2−2BraE2−3BraE2−4BraE2−5BraE2−6BraE2−7BraE2−8BraE2−9BraE2−10BraE2−11BraE2−12BraE2−13BraE2−14BraE2−15BraE2−16BraE2−17BraE2−18BraE2−19BraE2−20BraE2−21BraE2−22BraE2−23BraE2−24BraE2−25BraE2−26BraE2−27BraE2−28BraE2−29BraE2−30BraE2−31BraE2−32BraE2−33BraE2−34BraE2−35BraE2−36BraE2−37BraE2−38BraE2−39BraE2−40BraE2−41BraE2−42BraE2−43BraE2−44BraE2−45BraE2−46BraE2−47BraE2−48BraE2−49BraE2−50BraE2−51BraE2−52BraE2−53BraE2−54BraE2−55BraE2−56BraE2−57BraE2−59BraE2−60BraE2−61BraE2−62BraE2−63BraE2−64BraE2−65BraE2−66BraE2−67BraE2−68BraE2−69BraE2−70BraE2−71BraE2−72BraE2−73BraE2−74BraE2−75BraE2−76BraE2−77BraE2−80BraE2−82BraE2−83

−1.5

−1

−0.5

0

0.5

1

1.5

(a)



0

1

00

0

00

2

00

3

680

00

0

2

0

0

2

0

0

0

0

0

0

0

2

0 0 0Root

Stem

Leaf

Flower

Silique

(b)

Flower

Silique

Root

Stem

Leaf

BraE2−5−BraE2−7 (0.89671)BraE2−6−BraE2−12 (0.733762)BraE2−19−BraE2−68 (0.845815)BraE2−20−BraE2−21 (−0.04821)BraE2−24−BraE−25 (0.306023)BraE2−27−BraE2−29 (0.287456)BraE2−32−BraE2−33 (0.239577)BraE2−35−BraE2−69 (0.384983)BraE2−38−BraE2−39 (0.895816)BraE2−42−BraE2−44 (0.9584)BraE2−46−BraE2−48 (0.874904)BraE2−56−BraE2−57 (0.879441)BraE2−61−BraE2−62 (0.015553)BraE2−65−BraE2−66 (0.791393)BraE2−72−BraE2−73 (0.697466) BraE2−74−BraE2−75 (0.914105)BraE2−81−BraE2-82 (NA)BraE2−9−BraE2−31 (0.423102)BraE2−15−BraE2−16 (0.938003)BraE2−17−BraE2−18 (0.407923)BraE2−30−BraE2−34 (0.453099)BraE2−50−BraE2−51 (0.212499)BraE2−54−BraE2−55 (0.315854)BraE2−58−BraE2−59 (NA)BraE2−80−BraE2−83 (−0.09499)

−1.5

−1−0.5

00.5

11.5

(c)

Figure 5: (a) Heat map of expression profiles (in log2-based FPKM) for BraE2s in the five tissues of stem, root, leaf, flower, and silique. Theexpression levels are exhibited by the color bar. (b) Venn diagram analysis of the tissue expression of BraE2s. (c). Heat map of expressionprofiles for BraE2 25 paralogous pairs in the five tissues of stem, root, leaf, flower, and silique. The Pearson correlation coefficient (PCC) isalso displayed in bracket while NA indicate no available results for PCC.


34.87%

3.19%4.11%

17.92%

39.9%

Light (40)Hormones (13)Heat and Cold Stress (4)

CircadianOthers (30)

Cis-Elements

(a)BraE2-80

BraE2-46

BraE2-78

BraE2-54 BraE2-51BraE2-55 BraE2-56BraE2-57BraE2-24BraE2-42

BraE2-65BraE2-48


BraE2-25

BraE2-47BraE2-44

BraE2-77


BraE2-81


BraE2-39BraE2-72

BraE2-36BraE2-35



BraE2-21 BraE2-74BraE2-3 BraE2-83BraE2-68

BraE2-67BraE2-71

BraE2-60 BraE2-16BraE2-28BraE2-19 BraE2-61 BraE2-75BraE2-62BraE2-27

BraE2-12BraE2-30BraE2-14BraE2-63BraE2-41

BraE2-37BraE2-29 BraE2-82 BraE2-50BraE2-10 BraE2-33 BraE2-11BraE2-34


BraE2-40


BraE2-31BraE2-20BraE2-45 BraE2-43

BraE2-53

(b)



ABA

−1

ABA

−6

ABA

−12

GA

−1

GA

−6

GA

−12

JA−1

JA−6

JA−12

BraE2−5BraE2−7BraE2−6BraE2−12BraE2−19BraE2−68BraE2−38BraE2−39BraE2−42BraE2−44BraE2−46BraE2−48BraE2−56BraE2−57BraE2−65BraE2−66BraE2−72BraE2−73BraE2−74BraE2−75BraE2−9..BraE2−31..BraE2−17BraE2−18BraE2−30BraE2−34BraE2−54BraE2−55BraE2−35BraE2−69

BR−1

BR−6

BR−12


−1−0.5

00.5

1

-0.041816615

Pearson Correlation Pearson Correlation

-0.705792241

0.888133654

0.489588355

-0.601089733

0.902937678

-0.907015852

-0.454991538

0.689262032

0.416815188

0.998000705

-0.299771854

-0.745118139

-0.468633178

-0.187197738



0.987823455

-0.235665961

0.998902101

0.431151701

-0.093613694

0.961331638

-0.564193417

-0.194124848

0.284269363

0.975542587

0.422922623

-0.01740651

0.968219671

0.145773916

0.824723901

0.758434057

0.483593816

-0.454700738

0.89516653

0.997525287

0.981522316

-0.899164553

-0.433017463

-0.471792152

0.999269781

0.970592302

0.909069788

-0.996051544

0.889828906

-0.203389533

0.982214351

-0.486391286

0.046040844

0.137801637

0.747490152

0.595873614

0.3005184

0.999994207

-0.540940911

0.886988011

0.98816035

-0.224676354

0.998857926

0.59709217

0.455850939

A B

C D

(c)



Heat−1

Heat−6

Heat−12


NaC

l−1

NaC

l−6

NaC

l−12


Cold−1

Cold−6

Cold−12


−1−0.5

00.5

1

PEG−1

PEG−6

PEG−12


0.999469323

-0.531813281

0.990319165

-0.379240461

0.1108027

-0.817519792

-0.52835569

0.271191015

-0.286454536

0.99458637

-0.9763791

0.392901263

0.999993863

-0.952123899

-0.351480832

0.970778818

0.921465118

-0.09962587

0.315812107

-0.248353804

0.815619435

-0.961732163

0.881851377

0.999967954

-0.577131335

-0.274115307

0.98763818

0.936237083

0.152505889

0.703445291

0.960830814

-0.485480307

0.471525464

0.99902635

0.588995323

-0.998180782

0.974062562

0.582424302

-0.829617489

-0.412030444

0.089969222

-0.476646563

-0.839056302

-0.484137997

-0.621016742

0.832201329

0.99820801

0.505927784

0.324533747

-0.940791704

-0.061731902

-0.701231025

-0.217089694

0.66716027

-0.999923056

0.720350421

0.86447759

0.79130462

0.999852069

0.9775246

E F

HG

Pearson CorrelationPearson Correlation

(d)



ABAGAJABR

PEGNaClHeatCold

Up- regulated

Down- regulated

(e)

High

Medium

Negative 0

2

4

6

8

ABAGA

JABR

PEGNaCl

HeatCold

ABAGAJABR

PEGNaClHeatCold

PCC

Valu

es

Ranges Stresses

(f)

Figure 6: (a) Showing the ratio of different cis-element. (b) The coregulatory network for BraE2s was presented by STRING sever. (c and d)Expression analysis of the BraE2 genes under multiple abiotic and hormone treatments in Brassica rapa (A-H). Heat map representation ofthe BraE2 genes for abiotic/hormone treatments (namely, ABA, GA, JA, BR, PEG, NaCl, heat, and cold stress). Each pair of BraE2 along theirPCC values is also displayed. (e) Showing the up- and downregulated genes in response to multiple abiotic/hormone treatments. (f) Showingthe PCC values under response to multiple abiotic/phytohormones treatments.

they might have acquired new functions for plant improve-ment. However, there was divergence in the expressionpattern among the duplicated paralogous pairs which furthersuggested that after duplication in the evolutionary processsome of them may acquire new functions. To understandthe expression mechanisms of BraE2s, we identified commoncis-regulatory elements in the promoter regions of BraE2s.The results, provided valuable information as majority ofgenes, were involved in light regulation, hormones, and otherkey biotic-abiotic factors. Based on the results of RNA-seq data and cis-elements, we performed qRT-PCR aftermultiple abiotic and hormone treatments. We selected 11pairs of duplicated types and 4 pairs randomly among BraE2genes to explore the expression profile of BraE2. The ratiosfor majority of treatments were upregulated, such as forheat stress (55.55%), GA (52.22%), PEG, and NaCl stress(50%); however BR was more sensitive as a large number(74.44%) of genes were downregulated. Furthermore, basedon PCC values, cold stress was among the highest with (9)PCC values (>0.6) which signify a close relationship andtheir expression profiles resulted in (46.67%) upregulation ingenes. We further speculated that the function of genes wasenhanced and expanded through genes duplication. Though,functional analysis will confirm and determine the pivotalrole of BraE2. The expression levels of AhUBC2 in peanutplants were regulated by PEG, NaCl, ABA, and physiologicalstress [18]. In response to biotic-abiotic stresses and cellular

responses from wild rice, OgUBC1 were reported [35]. InArabidopsis, through application of salt stress AtUBC32were greatly influenced and were reported to play a role inthe BR (brassinosteroid) salt stress tolerant plant [19]. Inplants, response to abiotic stresses and hormone-signaling areintegral and it has been reported that in hormone-mediatedstress responses the E2s play a major role [3–5]. These resultspeculated that E2s are critical for multiple stress responsesin various species. Therefore, taken together, our resultsspeculated that BraE2 genes family might be contributinginto functional divergence and playing a critical role duringabiotic/hormone stresses.

5. Conclusion

In this study, a total of 83 members of BraE2 were identifiedand were classified into four major classes based on thedomain information and phylogenetic tree. To predict thefunctional characteristics of BraE2, we analyzed the physic-ochemical properties along with gene duplication analysis.RNA-seq data and qRT-PCR results presented significantroles of BraE2 in the growth, development, and resistancemechanism of B. rapa under various stresses. The intron-exon distribution for most of the genes shared a patternof junction, and gene duplications analysis showed thatsegmental duplication being major factor for the expansionand close evolutionary relationships of the gene family. Based


on our results, as evidenced by gene expression analysisespecially under various abiotic and hormone stresses, wehypothesize that BraE2 genes show divergence in theirfunction. Moreover, these results provide novel insight byproviding a solid foundation for future functional dissectionof BraE2 family in B. rapa.

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed a potential conflicts of interest.

Authors’ Contributions

Nadeem Khan, Chun-mei Hu, and Waleed Amjad Khanconceived and designed the experiments. Nadeem Khanand Emal Naseri performed the experiments. Nadeem Khananalyzed the data and wrote the manuscript. Han Ke, DongHuijie, and Xilin Hou helped in revision. All authors read andapproved the manuscript.

Acknowledgments

The authors express their gratitude to Dr. Badshah Islamand Dr. Amjad Iqbal for helpful and serious comments toimprove the paper. This study was financially supported bythe Independent Innovation Fund for Agricultural Scienceand Technology of Jiangsu Province (CX (15) 1015) and NorthJiangsu Special Project of Jiangsu Province (SZ-LYG2017027).

Supplementary Materials

Supplementary 1. Figure. Is. Showing the PCC values for mul-tiple treatments with values > 0.5.Supplementary 2. Table. 1. Sequences of the BraE2 gene prim-ers used for quantitative real-time PCR.Supplementary 3. Table. 2. The basic description of BraE2genes in Brassica rapa.Supplementary 4. Table. 3. Identification of BraE2 syntenicgenes between A. thaliana along with three subgenomes ofBrassica rapa.Supplementary 5. Table. 4. The gene with outlier Ka/Ks val-ues and estimated divergence time for syntenic pairs of BraE2.Supplementary 6. Table. 5. The FPKMvalues of BraE2 genes.Supplementary 7. Table. 6. Syntenic paralog pair of BraE2 withPC and FPKM values.Supplementary 8. Table. 7. Cis-elements of BraE2 genes inBrassica rapa.

Supplementary 9. Table. 8. Relative expression pattern ofBraE2 genes along with PC values with respect to variousstresses by qRT-PCR.Supplementary 10. Table. 9. Pearson correlation coefficient ofthe stress-induced BraE2 genes whose PC is greater than 0.5.

References

[1] B. T. Dye and B. A. Schulman, “Structural mechanisms under-lying posttranslational modification by ubiquitin-like proteins,”Annual Review of Biophysics, vol. 36, pp. 131–150, 2007.

[2] R. D. Vierstra, “Proteolysis in plants: mechanisms and func-tions,” in Post-Transcriptional Control of Gene Expression inPlants, W. Filipowicz and T. Hohn, Eds., pp. 275–302, Springer,Dordrecht, Netherlands, 1996.

[3] R. L. Welchman, C. Gordon, and R. J. Mayer, “Ubiquitinand ubiquitin-like proteins as multifunctional signals,” NatureReviews Molecular Cell Biology, vol. 6, no. 8, pp. 599–609, 2005.

[4] A. Sadanandom, M. Bailey, R. Ewan, J. Lee, and S. Nelis,“The ubiquitin-proteasome system: Central modifier of plantsignalling,” New Phytologist, vol. 196, no. 1, pp. 13–28, 2012.

[5] K. Dreher and J. Callis, “Ubiquitin, hormones and biotic stressin plants,” Annals of Botany, vol. 99, no. 5, pp. 787–822, 2007.

[6] A. Hershko, H. Heller, S. Elias, and A. Ciechanover, “Com-ponents of ubiquitin-protein ligase system. Resolution, affinitypurification, and role in protein breakdown,” The Journal ofBiological Chemistry, vol. 258, no. 13, pp. 8206–8214, 1983.

[7] Y. Gao, Y. Wang, H. Xin, S. Li, and Z. Liang, “Involvement ofUbiquitin-Conjugating Enzyme (E2 Gene Family) in RipeningProcess and Response to Cold and Heat Stress of Vitis vinifera,”Scientific Reports, vol. 7, no. 1, 2017.

[8] B. Sharma and T. K. Bhatt, “Genome-wide identification andexpression analysis of E2 ubiquitin-conjugating enzymes intomato,” Scientific Reports, vol. 7, no. 1, 2017.

[9] Z. E, Y. Zhang, T. Li, L. Wang, H. Zhao, and G. K. Pandey,“Characterization of the Ubiquitin-Conjugating Enzyme GeneFamily in Rice and Evaluation of Expression Profiles underAbiotic Stresses and Hormone Treatments,” PLoS ONE, vol. 10,no. 4, p. e0122621, 2015.

[10] D. Jue, X. Sang, S. Lu et al., “Genome-wide identification, phy-logenetic and expression analyses of the ubiquitin- conjugatingenzyme gene family in Maize,” PLoS ONE, vol. 10, no. 11, 2015.

[11] D. Jue, X. Sang, B. Shu et al., “Characterization and expressionanalysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya,” PLoS ONE, vol. 12, no.2, 2017.

[12] E. Kraft, S. L. Stone, L. Ma et al., “Genome analysis andfunctional characterization of the E2 and RING-type E3 ligaseubiquitination enzymes of Arabidopsis,” Plant Physiology, vol.139, no. 4, pp. 1597–1611, 2005.

[13] M. Zhen, R. Heinlein, D. Jones, S. Jentsch, and E. P.M. Candido,“Theubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation,”Molecular and Cellular Biology, vol. 13, no. 3, pp. 1371–1377, 1993.

[14] S. J. L. Van Wijk and H. T. M. Timmers, “The family ofubiquitin-conjugating enzymes (E2s): Deciding between lifeand death of proteins,” The FASEB Journal, vol. 24, no. 4, pp.981–993, 2010.

[15] C. A. Semple, “The Comparative Proteomics of UbiquitinationinMouse,”Genome Research, vol. 13, no. 6, pp. 1389–1394, 2003.

http://downloads.hindawi.com/journals/bmri/2018/5206758.f1.pdf

http://downloads.hindawi.com/journals/bmri/2018/5206758.f2.xlsx










[16] S. Yang, J. R. Arguello, X. Li et al., “Repetitive element-mediatedrecombination as a mechanism for new gene origination inDrosophila,” PLoS Genetics, vol. 4, no. 1, pp. 0078–0087, 2008.

[17] G.-A. Zhou, R.-Z. Chang, and L.-J. Qiu, “Overexpression ofsoybean ubiquitin-conjugating enzyme gene GmUBC2 confersenhanced drought and salt tolerance through modulating abi-otic stress-responsive gene expression in Arabidopsis,” PlantMolecular Biology, vol. 72, no. 4, pp. 357–367, 2010.

[18] X. Wan, A. Mo, S. Liu, L. Yang, and L. Li, “Constitutiveexpression of a peanut ubiquitin-conjugating enzyme gene inArabidopsis confers improved water-stress tolerance throughregulation of stress-responsive gene expression,” Journal ofBioscience and Bioengineering, vol. 111, no. 4, pp. 478–484, 2011.

[19] F. Cui, L. Liu, Q. Zhao et al., “Arabidopsis ubiquitin con-jugase UBC32 is an ERAD component that functions inbrassinosteroid-mediated salt stress tolerance,” The Plant Cell,vol. 24, no. 1, pp. 233–244, 2012.

[20] E. Chung, C.-W.Cho,H.-A. So, J.-S. Kang, Y. S. Chung, and J.-H.Lee, “Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance inArabidopsis,” PLoS ONE, vol. 8, no. 6, 2013.

[21] L. Xu, R. Menard, A. Berr et al., “The E2 ubiquitin-conjugatingenzymes, AtUBC1 and AtUBC2, play redundant roles and areinvolved in activation of FLC expression and repression offlowering inArabidopsis thaliana,”ThePlant Journal, vol. 57, no.2, pp. 279–288, 2009.

[22] C. D. Town, F. Cheung, R. Maiti et al., “Comparative genomicsof Brassica oleracea and Arabidopsis thaliana reveal gene loss,fragmentation, and dispersal after polyploidy,” The Plant Cell,vol. 18, no. 6, pp. 1348–1359, 2006.

[23] The Brassica rapa Genome Sequencing Project Consortium, X.Wang, H. Wang et al., “The genome of the mesopolyploid cropspecies Brassica rapa,” Nature Genetics, vol. 43, pp. 1035–1039,2011.

[24] S. Kumar, G. Stecher, and K. Tamura, “MEGA7: MolecularEvolutionary Genetics Analysis version 7.0 for bigger datasets,”Molecular Biology and Evolution, vol. 33, no. 7, pp. 1870–1874,2016.

[25] R. E. Voorrips, “Mapchart: software for the graphical presenta-tion of linkage maps and QTLs,” Journal of Heredity, vol. 93, no.1, pp. 77-78, 2002.

[26] S. Rombauts, P. Dehais, M. Van Montagu, and P. Rouze,“PlantCARE, a plant cis-acting regulatory element database,”Nucleic Acids Research, vol. 27, no. 1, pp. 295-296, 1999.

[27] C. Tong, X.Wang, J. Yu et al., “Comprehensive analysis of RNA-seq data reveals the complexity of the transcriptome in Brassicarapa,” BMC Genomics, vol. 14, no. 1, article no. 689, 2013.

[28] C. A. Heid, J. Stevens, K. J. Livak, and P.M.Williams, “Real timequantitative PCR,”Genome Research, vol. 6, no. 10, pp. 986–994,1996.

[29] N. Khan, C.-m. Hu, W. A. Khan, and X. Hou, “Genome-WideIdentification, Classification, and Expression Divergence ofGlutathione-Transferase Family inBrassica rapa underMultipleHormone Treatments,” BioMed Research International, vol.2018, Article ID 6023457, 19 pages, 2018.

[30] E. Papaleo, N. Casiraghi, A. Arrigoni, M. Vanoni, P. Coccetti,and L. de Gioia, “Loop 7 of E2 enzymes: An ancestral conservedfunctional motif involved in the E2-mediated steps of theubiquitination cascade,” PLoS ONE, vol. 7, no. 7, 2012.

[31] F.-R. Schumacher, G. Wilson, and C. L. Day, “The N-terminalextension of UBE2E ubiquitin-conjugating enzymes limits

Chain assembly,” Journal of Molecular Biology, vol. 425, no. 22,pp. 4099–4111, 2013.

[32] M. Lynch and J. S. Conery, “The evolutionary fate and conse-quences of duplicate genes,” Science, vol. 290, no. 5494, pp. 1151–1155, 2000.

[33] T. Tohge and A. R. Fernie, “Combining genetic diversity,informatics and metabolomics to facilitate annotation of plantgene function.,” Nature Protocols, vol. 5, no. 6, pp. 1210–1227,2010.

[34] H. Ma and J. Zhao, “Genome-wide identification, classification,and expression analysis of the arabinogalactan protein genefamily in rice (Oryza sativa L.),” Journal of Experimental Botany,vol. 61, no. 10, pp. 2647–2668, 2010.

[35] E. H. Jeon, J. H. Pak, M. J. Kim et al., “Ectopic expression ofubiquitin-conjugating enzyme gene from wild rice, OgUBC1,confers resistance againstUV-B radiation andBotrytis infectionin Arabidopsis thaliana,” Biochemical and Biophysical ResearchCommunications, vol. 427, no. 2, pp. 309–314, 2012.

[36] Y.-H. Jiang and A. L. Beaudet, “Human disorders of ubiq-uitination and proteasomal degradation,” Current Opinion inPediatrics, vol. 16, no. 4, pp. 419–426, 2004.

[37] G. Xu, C. Guo, H. Shan, and H. Kong, “Divergence of duplicategenes in exon-intron structure,” Proceedings of the NationalAcadamy of Sciences of the United States of America, vol. 109, no.4, pp. 1187–1192, 2012.

[38] J. A. Birchler and R. A. Veitia, “The gene balance hypothesis:from classical genetics tomodern genomics,”ThePlant Cell, vol.19, no. 2, pp. 395–402, 2007.

[39] J. Ziarovska, M. Zahorsky, Z. Galova, and A. Hricova, “Bioin-formatic approach in the identification of Arabidopsis genehomologous in Amaranthus,” Potravinarstvo, vol. 9, no. 1, pp.149–153, 2015.

[40] E. W. Ganko, B. C. Meyers, and T. J. Vision, “Divergence inexpression between duplicated genes inArabidopsis,”MolecularBiology and Evolution, vol. 24, no. 10, pp. 2298–2309, 2007.

[41] W.-H. Li, J. Yang, and X. Gu, “Expression divergence betweenduplicate genes,” Trends in Genetics, vol. 21, no. 11, pp. 602–607,2005.

[42] J. Huerta-Cepas, J. Dopazo, M. A. Huynen, and T. Gabaldon,“Evidence for short-time divergence and long-time conser-vation of tissue-specific expression after gene duplication,”Briefings in Bioinformatics, vol. 12, no. 5, pp. 442–448, 2011.

Hindawiwww.hindawi.com

International Journal of

Volume 2018

Zoology

Hindawiwww.hindawi.com Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation http://www.hindawi.com Volume 2013Hindawiwww.hindawi.com

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwww.hindawi.com Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research


International Journal of

MicrobiologyHindawiwww.hindawi.com

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwww.hindawi.com

https://www.hindawi.com/journals/ijz/

https://www.hindawi.com/journals/ari/

https://www.hindawi.com/journals/ijpep/

https://www.hindawi.com/journals/jpr/

https://www.hindawi.com/journals/ijg/

https://www.hindawi.com/journals/tswj/

https://www.hindawi.com/journals/abi/

https://www.hindawi.com/journals/jmb/

https://www.hindawi.com/journals/neuroscience/

https://www.hindawi.com/journals/bmri/

https://www.hindawi.com/journals/ijcb/

https://www.hindawi.com/journals/bri/

https://www.hindawi.com/journals/archaea/

https://www.hindawi.com/journals/gri/

https://www.hindawi.com/journals/av/

https://www.hindawi.com/journals/sci/

https://www.hindawi.com/journals/er/

https://www.hindawi.com/journals/ijmicro/

https://www.hindawi.com/journals/jna/

https://www.hindawi.com/

https://www.hindawi.com/

Evolution and Expression Divergence of E2 Gene Family...

Documents

Transcript of Evolution and Expression Divergence of E2 Gene Family...