Mol Genet Genomics (2014) 289:77–91 DOI 10.1007/s00438-013-0791-3

ORIGINAL PAPER

Genome‑wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis) Xiao‑Ming Song · Zhi‑Nan Huang · Wei‑Ke Duan · Jun Ren · Tong‑Kun Liu · Ying Li · Xi‑Lin Hou 

Received: 14 August 2013 / Accepted: 31 October 2013 / Published online: 17 November 2013 © Springer-Verlag Berlin Heidelberg 2013

Abstract  Basic helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotic organisms and are thought to be one of the largest families of regulatory proteins. This important family of transcriptional regulators plays crucial roles in plant development. However, a systematic analysis of the bHLH transcription factor family has not been reported in Chinese cabbage. In this study, 230 bHLH transcription factors were identified from the whole Chinese cabbage genome and compared with proteins from other representative plants, fungi and metazoans. The Chinese cabbage bHLH (BrabHLH) gene family could be classified into 24 subfamilies. Phylogenetic analysis of BrabHLHs along with bHLHs from Arabidopsis and rice indicated 26 subfamilies. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns and interaction networks of BrabHLHs were analyzed. Distribution mapping showed that BrabHLHs were non-randomly located on the ten Chinese cabbage chromosomes. One hundred and twenty-four orthologous bHLH genes were identified between Chinese cabbage and Arabidopsis, and the interaction networks of the orthologous Communicated by Y. Van de Peer. Electronic supplementary material  The online version of this article (doi:10.1007/s00438-013-0791-3) contains supplementary material, which is available to authorized users. X.-M. Song · Z.-N. Huang · W.-K. Duan · J. Ren · T.-K. Liu · Y. Li · X.-L. Hou (*)  State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected]

genes were constructed in Chinese cabbage. Quantitative RT-PCR analysis showed that expressions of BrabHLH genes varied widely under different abiotic stress treatments for different times. Thus, this comprehensive analysis of BrabHLHs represents a rich resource, aiding the elucidation of the roles of bHLH family members in plant growth and development. Furthermore, the comparative genomics analysis deepened our understanding of the evolution of this gene family after a polyploidy event. Keywords Genome-wide analysis · bHLH transcription factor · Abiotic stress · qRT-PCR · Chinese cabbage

Introduction Crops of the genus Brassica are widely used for vegetables, oilseed, condiments and fodder. Asia currently accounts for 61 % of the yield of Brassica (http://faostat.fao.org). Brassica includes B. rapa, which comprises several subspecies, such as Chinese cabbage (B. rapa ssp. pekinensis), nonheading Chinese cabbage (B. rapa ssp. chinensis) and turnip (B. rapa ssp. rapifera). Chinese cabbage is one of the most important vegetables in Asia. As the region of origin of Chinese cabbage, China has rich and diverse germplasm resources. Chinese cabbage has experienced thousands of years of cultivation and artificial selection. Given the important economic value and its close relationship with Arabidopsis, the Chinese cabbage (Chiifu-401-42) genome has been sequenced and assembled (Wang et al. 2011). The whole Chinese cabbage genome sequences and more than 40,000 proteins have been obtained. The genome has undergone triplication events since its divergence from Arabidopsis (13–17 mya) (Beilstein et al. 2010; Wang et al. 2011); however, a high degree of sequence similarity and

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conserved genome structure remain between the two species. Arabidopsis is, therefore, a viable reference species for comparative genomics studies. Variation in the number of members in gene families caused by genome triplication may contribute to the broad range of phenotypic plasticity and increased tolerance to environmental extremes observed in Brassica species. The bHLH transcription factors are important regulators involved in plant developmental and physiological processes. There have been few reports about bHLH proteins in Chinese cabbage. Therefore, it is appropriate to analyze bHLH genes in the whole genome. The bHLH proteins contain approximately 50–60 amino acids, with two functionally distinctive segments, the basic region and the HLH region. The basic region, located at the N-terminus, is involved in DNA binding and comprises about 15 amino acids, which typically include six basic residues (Atchley et al. 1999; Li et al. 2006). The HLH region contains two amphipathic α-helices separated by a loop region of variable length or sequence (ToledoOrtiz et al. 2003) and functions as a dimerization domain that can form homodimers or heterodimers by interacting with other bHLH proteins (Murre et al. 1989). Among all the bHLH motifs, 19 amino acids have been found to be highly conserved in organisms ranging from yeast to mammals (Wang et al. 2009b; Zheng et al. 2009). Outside of the conserved bHLH domain, these proteins exhibit considerable sequence divergence (Atchley et al. 1999). Many bHLH transcription factors have been identified in the completed genome sequences of over 20 organisms, such as Saccharomyces cerevisiae, Homo sapiens, Arabidopsis thaliana and Oryza sativa (Dang et al. 2011). In early research, the bHLH transcription factor family was divided into six groups (A–F) in metazoans, which contained 45 subfamilies based on their different functions in the regulation of gene expression, different target DNA elements and phylogenetic analyses (Atchley et al. 1999; Simionato et al. 2007; Liu and Zhao 2010). Group A contained 22 subfamilies that bind to CACCTG or CAGCTG core sequences of E boxes, and their functions were the regulation of sex determination, trophoblast cell development and mesoderm formation. Group B consisted of 12 subfamilies that bind to CACGTG or CATGTTG core sequences of E boxes. They mainly controlled the expression of glucose-responsive genes and regulated the sterol metabolism. Group C had seven subfamilies, and they were closely associated with the regulation of midline and tracheal development. They not only had a bHLH domain, but also contained a PAS domain, binding to ACGTG or GCGTG sequences. Group D had only one subfamily and they lack a basic domain. They exerted their functions by formatting heterodimers with group A proteins. Group E contained two subfamilies, which bind preferentially to

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Mol Genet Genomics (2014) 289:77–91

typical sequences of N boxes (CACGCG or CACGAG), and they regulated embryonic segmentation, somitogenesis and organogenesis. Group F proteins have the COE domain, which also has an additional domain involved in both dimerization and DNA binding. The protein functions of this group were mainly regulation of head development and formation of olfactory sensory neurons (Atchley and Fitch 1997; Ledent and Vervoort 2001; Ledent et al. 2002). Compared with metazoans, the analyses of the bHLH proteins were mainly conducted in model plants, and only a small number of plant bHLH protein functions have been reported. An early study showed that there were 133 bHLH transcription factors in Arabidopsis (Heim et al. 2003). With the completion of whole genome sequences of model plants Arabidopsis and rice, 147 bHLH transcription factors were identified in Arabidopsis, which were divided into 21 subfamilies according to phylogenetic analyses (Toledo-Ortiz et al. 2003). One hundred and sixty-seven bHLH genes have been identified in the rice genome (Li et al. 2006). Using whole genome sequences, the relationships of bHLH proteins among nine species of land plants and algae were analyzed. Phylogenetic analyses showed that the plants bHLH proteins comprised 26 subfamilies, 20 of which were present in the common ancestors of extant mosses and vascular plants, which are thought to have existed 440 million years ago (Pires and Dolan 2010). In metazoans, phylogenetic analyses demonstrated the presence of at least 44 subfamilies in the common ancestor (Simionato et al. 2007). Furthermore, studies showed that the ancestor of metazoans for bHLH proteins was present 600–700 million years ago, which was earlier than that of plants (Douzery et al. 2004; Peterson et al. 2004). A phylogenetic tree of seven species was constructed to analyze the function and ancestral relationships of all 541 identified bHLH proteins (Stevens et al. 2008). A previous study indicated that group A primarily contained mammalian proteins and lacked plant bHLH proteins, while group F only contained plant bHLH proteins. The other four groups had a mixture of different species bHLH proteins, and most plant bHLH proteins belonged to group B (Stevens et al. 2008; Skinner et al. 2010). However, these analyses were only based on a small number of plants and metazoans. Recently, the genomes of many species have been completely sequenced, which provides an opportunity to gain a deeper insight into the bHLH proteins using comparative genomics among large numbers of plants and metazoans. Furthermore, the wealth of functional annotations provides us with the opportunity to use a phylogenetic tree to predict potential functions of uncharacterized members in the same subfamily. In this study, we systematically and comprehensively describe the bHLH transcription factors in Chinese cabbage through a comparative genomic analysis. The objectives

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of our study were as follows: (1) to identify and map the bHLH transcription factors on to the ten Chinese cabbage chromosomes; (2) to analyze bHLH transcription factor phylogenetic relationships among plants, metazoans and fungi using comparative genomics; (3) to construct bHLH transcription factor interaction networks using the orthologous and paralogous bHLH proteins identified in Chinese cabbage and Arabidopsis and rice; and (4) to carry out bHLH transcription factor expression analysis under various abiotic stress treatments using qRT-PCR. This study provided a useful resource for future studies on the structure and function of bHLH proteins in the regulation of Chinese cabbage development. The results also provide a method for identifying and characterizing bHLH transcription factors in other species.

Materials and methods Sequence retrieval The A. thaliana bHLH proteins were retrieved from TAIR (http://www.arabidopsis.org/) according to previous reports (Heim et al. 2003; Toledo-Ortiz et al. 2003; Pires and Dolan 2010). A clear bHLH domain was not found in At5g64340 (AtbHLH142), At5g50010 (AtbHLH145), At4g30180 (Atb HLH146), At3g17100 (AtbHLH147), At3g05800 (AtbHLH 150), At2g47270 (AtbHLH151), At1g22380 (AtbHLH152), At1g64625 (AtbHLH157), At2g43060 (AtbHLH158) and At4g30410 (AtbHLH159); therefore, they were excluded from the study. The data set of predicted O. sativa bHLH proteins was retrieved from previous analyses (Li et al. 2006; Pires and Dolan 2010). A bHLH domain was not found in Os01g65080 (OsbHLH033), Os06g41060 (OsbHLH095), Os07g08440 (OsbHLH099), Os04g35000 (OsbHLH145), Os02g08220 (OsbHLH157), Os11g02054 (OsbHLH160) and Os12g02020 (OsbHLH161). The bHLH proteins of Selaginella moellendorffii, Ostreococcus tauri, Chlorella vulgaris and Saccharomyces cerevisiae were retrieved from a previous report (Pires and Dolan 2010). The data set of predicted Zea mays, Vitis vinifera, Sorghum bicolor, Populus trichocarpa, Medicago truncatula, Glycine max, Cucumis sativus, Carica papaya, Brachypodium distachyon and Physcomitrella patens bHLH proteins was downloaded from the Plant Transcription Factor Database (http://planttfdb.cbi.edu.cn/index.php, v2.0) (Zhang et al. 2011). The Pfam database (http://pfam.sanger.ac.uk/) (Punta et al. 2012) was used to screen the genome assemblies of Brassica rapa (http://brassicadb.org/brad/), Solanum lycopersicum (http://solgenomics.net/organism/Solanum_lycopersicum/ genome), Solanum tuberosum (http://solanaceae.plantbiolo gy.msu.edu/pgsc_download.shtml), Musa acuminata (http://

banana-genome.cirad.fr/download.php), Citrus sinensis (http://citrus.hzau.edu.cn/orange), Malus x domestica (http:// genomics.research.iasma.it/), Theobroma cacao (http://coco agendb.cirad.fr/gbrowse/download.html), Chlamydomonas reinhardtii (http://www.phytozome.net), Cyanidioschyzon merolae (http://merolae.biol.s.u-tokyo.ac.jp/) and Volvox carteri (http://www.plantgdb.org/XGDB/phplib/download.php). The bHLH proteins of 12 different species, representative of the main metazoans lineages, were obtained from a previous report (Simionato et al. 2007). Phylogenetic analysis and identification of conserved motifs The complete amino acid sequences were screened against the Pfam database to identify the domains of bHLH transcription factors. MEGA5 software (Tamura et al. 2011) was used to construct neighbor-joining (NJ) distance trees using Chinese cabbage bHLH protein domain sequences and those from Arabidopsis and rice. The bootstrap was set as 1,000 replicates, which provided information about their statistical reliability. Furthermore, a phylogenetic tree of all the identified bHLH protein domains was also constructed. To construct the NJ distance trees, 3,242 plant, 698 metazoan and eight fungal bHLH domains were used. The identified bHLH domains were aligned using ClustalX 2.0 program with default settings (Thompson et al. 1997). The aligned bHLH domains were checked manually for their amino acid residues at the conserved sites, which were shaded by DNAMAN software (http: //www.lynnon.com/). Perl scripts were used to obtain the intron and exon structure of bHLH domain protein genes. MEME software (http://meme.sdsc.edu/meme/) was used to search for conserved motifs in the complete amino acid sequences of bHLH proteins (Bailey et al. 2009). The location of bHLH genes on chromosomes and identification of orthologous and paralogous genes The position of each BrabHLH gene on the ten chromosomes was obtained from the BRAD site (http://brassicadb.org/brad/), and was marked on the chromosome using a Perl script. The orthologous and paralogous bHLH genes in Chinese cabbage, Arabidopsis and rice were identified using OrthoMCL (http://www.orthomcl.org/cgi-bin/OrthoMclWeb.cgi) (Li et al. 2003). The relationships of orthologous and paralogous genes in these three species were shown using the Circos program (Krzywinski et al. 2009). The interaction network associated with Arabidopsis orthologous of bHLH genes in Chinese cabbage was constructed using the Arabidopsis interaction viewer and cytoscape software (Shannon et al. 2003). The bHLH genes in Chinese cabbage were

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searched for duplication events (e value 90 %).

Mol Genet Genomics (2014) 289:77–91

bonsai.hgc.jp/~mdehoon/software/cluster/software.htm), and the results were shown using Tree View software (http://jtreeview.sourceforge.net/).

Plant materials, growth conditions and stress treatments The Chinese cabbage cultivar Chiifu-401-42 was used for the experiment. Its complete genome has been sequenced as a typical cultivar for Chinese cabbage research. Seeds were grown in pots containing soil: vermiculite mixture (3:1) in a controlled-environment growth chamber programmed for 16/8 h at 25/20 °C for day/night, relative humidity of 55–60 %. One month later, seedlings at the five-leaf stage were transferred to growth chambers set at 4 or 38 °C, as the cold and heat treatments, under the same light intensity and day length and at the same time for acclimation. Some plants were cultured in 1/2 Hoagland’s solution in plastic containers, pH 6.5. After 5 days of acclimatization, plants were cultured in the following treatments: (1) control; (2) 100 μM abscisic acid (ABA); (3) 100 μM gibberellic acid (GA); and (3) polyethylene glycol (PEG) 6000 (10 %). The young leaf samples were collected at 4 and 12 h after these treatments. The control and stress-treated plants were harvested, frozen in liquid nitrogen and stored at −70 °C for further analysis. RNA isolation and quantitative real‑time PCR analysis Total RNA was isolated from plant leaves by an RNA kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. The RNAs were reverse transcribed into cDNA using the Prime Script RT reagent kit (TaKaRa, Kyoto, Japan). The Chinese cabbage actin gene (Bra028615) was used as an internal control to normalize the expression level of the target gene among different samples. The specific primers were designed according to the bHLH gene sequences using Primer 5.0 software (Supplementary Table 1). The qRT-PCR assays were performed with three biological and three technical replicates. Each reaction was performed in a 20-μL reaction mixture containing diluted cDNA sample as the template, SYBR Premix Ex Taq (2×) (TaKaRa, Kyoto, Japan) and gene specific primers. Quantitative real-time PCR was performed using MyiQ Single color Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA) with the following cycling profile: 94 °C for 30 s; followed by 40 cycles at 94 °C for 10 s, 58 °C for 30 s; and then a melting curve (61 cycles at 65 °C for 10 s) was generated to check the specific the amplification. The comparative Ct value method was employed to analyze the relative gene expression level. RNA level was expressed relative to the actin gene expression level as 2−∆∆CT, according to previous analyses (Pfaffl 2001). The bHLH genes expression cluster from each stress treatment was analyzed via the Cluster program (http://

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Results Identification of bHLH proteins in Chinese cabbage and comparative analyses Our search for bHLH-domain containing proteins identified 230 distinct bHLH transcription factors (Supplementary Table 2). For comparative genomic analyses, we searched for bHLH protein coding sequences in the representative genomes of 25 plants, 12 metazoans and one fungus (Fig. 1). Finally, a total of 3,242, 698 and eight bHLH proteins were identified in the plants, metazoans and fungi, respectively. These proteins represent the major evolutionary lineages of the species for the analysis of the bHLH transcription factors. The number of bHLH transcription factors in many higher plants was more than that in metazoans; in lower plants it was less than that in metazoans. Higher plants and metazoans have relatively more bHLH proteins than other eukaryotic species. This indicated that the increase in the number of bHLH proteins occurred independently during the evolution of plants and metazoans. Cumulatively, the number of bHLH transcription factors in Chinese cabbage (230) exceeded that in metazoans, fungi and most plants in our analyses. However, it was less than that in Musa acuminata (252), and Glycine max and Zea mays (289). In terms of the density of bHLH proteins in the whole Chinese cabbage genome (0.810), we found that it was more than that in all species used in our analyses, except Arabidopsis thaliana (1.111). Although there were more bHLH proteins in Glycine max and Zea mays than in Chinese cabbage, their bHLH protein densities (Glycine max, 0.296; Zea mays, 0.140) were lower than that in Chinese cabbage because of their large genome sizes. In the plants, we noted that the densities of bHLH proteins in all the higher plant genomes were more than those in all the lower plant genomes. This suggested that the bHLH proteins might play a very important role in plant evolution. Thus, the number and density of bHLH proteins increased as plants evolved, possibly because of genome duplication. Multiple sequence alignments, predicted DNA‑binding ability and conserved residues To analyze the sequence features of the BrabHLH domains, we performed multiple sequence alignment of 230 BrabHLH domain sequences (Supplementary Fig. 1). There were four conserved regions in the bHLH domain sequences, including one basic region, two helix regions

81

Mol Genet Genomics (2014) 289:77–91 Species Rosaceae

697.6

0.204

289

2066.4

0.140

Brachypodium distachyon

121

272.0

0.445

Oryza sativa

171

372.0

0.460

Musa acuminata

252

331.8

0.760

Selaginella moellendorffii

98

212.5

0.461

108

480.0

0.225

3

125.4

0.024

11

111.0

0.099

4 1 1

49.1 12.0 16.5

0.081 0.083 0.061

Saccharomyces cerevisiae

8

12.2

0.656

Drosophila melanogaster

58

176.0

0.330

Tribolium castaneum

54

205.4

0.263

Daphnia pulex

57

224.9

0.253

Caenorhabditis elegans

42

97.8

0.429

Mollusks

Lottia gigantea

63

420.5

0.150

Annelids

Capitella sp I

64

234.7

0.273

Homo sapiens Branchiostoma floridae Strongylocentrotus purpuratus Nematostella vectensis Hydra magnipapillata Amphimedon queenslandica

118 78 50 68 33 16

3423.0 575.0 870.4 224.9 1260.3 167.0

0.034 0.136 0.057 0.302 0.026 0.096

Brassicaceae Brassicales

Caricaceae

Sterculiaceae Rutaceae Vitaceae Lycopersicon Solanum

Angiosperms

Poaceae Monocotyledoneae

Higher plant Lycophytes Bryophyta

Plantae

Physcomitrella patens Chlorophyceae

Chlorophyta Lower plant

Trebouxiophyceae Prasinophyceae

Rhodophyta

Fungi

Arthropods Ecdysozoans Nematodes

Protostomes Trochozoans Bilaterians

Chordates Eumetazoans

Deuterostomes

Echinoderms

Metazoans

Zea mays

Number/Mb

142

Salicaceae

Vascular plant

Sorghum bicolor

Genome size (Mb)

0.219 0.527 0.384 0.296 0.385 1.111 0.810 0.652 0.309 0.420 0.398 0.184 0.175

Cucurbitaceae

Solanaceae

193 107 99 289 163 150 230 88 107 154 194 140 127

881.3 203.0 257.6 975.0 422.9 135.0 283.8 135.0 346.0 367.0 487.0 760.0 727.0

Fabaceae

Dicotyledoneae

Number of bHLH

Malus x domestica Cucumis sativus Medicago truncatula Glycine max Populus trichocarpa Arabidopsis thaliana Brassica rapa Carica papaya Theobroma cacao Citrus sinensis Vitis vinifera Solanum lycopersicum Solanum tuberosum

Cnidarians Sponges

Volvox carteri Chlamydomonas reinhardtii Chlorella vulgaris Ostreococcus tauri Cyanidioschyzon merolae

Fig. 1  Summary of bHLH transcription family factors of plants, metazoans and fungi

and one loop region. Generally, the conservation of the basic region and two helix regions was higher than that of the loop region. Most of the BrabHLH domains (171, 74.3 %) have five basic residues in the basic region, even though nine of these BrabHLH domains did not have the basic region. From the alignment, we identified 19 residues that are identical in at least 50 % of the 230 Chinese cabbage bHLH domains. Among these 19 residues, eight residues were present in more than 75 % sequences and Leu25 (L) in the first helix region was present in 100 %. Among the 19 conserved residues, five (His-5, Glu-9, Arg-10, Arg-12, Arg-13), five (Ile-18, Asn-19, Leu-25, Leu-28, Pro-30), two (Lys-39, Asp-45) and seven (Ala47, Leu-51, Ala-54, Ile-55, Tyr-57, Lys-59 Leu-61) amino acids were included in the basic region, the first helix region, the loop region and the second helix region, respectively. All of these conserved residues were consistent with previous reports (Atchley et al. 1999; Toledo-Ortiz et al. 2003). The identity of His-5 as a conserved residue, which was located in the basic region, was similar in Chinese cabbage and Arabidopsis in more than 50 % of sequences, but was different in rice (Supplementary Figs. 2 and 3). The basic region of the bHLH domain has the ability to bind to DNA and is critical for function. The BrabHLH

proteins were divided into several categories according to the sequences of the N-terminal region of their bHLH domains. The distribution of the predicted DNA-binding properties, as described below, was indicated with different colored lines on the ten chromosomes (Fig. 2). As well as Arabidopsis and rice, the BrabHLH proteins were also divided into two major groups according to the 17 N-terminal amino acids within the bHLH domain, including 196 DNA-binding proteins and 34 non-DNA-binding proteins. The DNA-binding bHLHs were further divided into two groups with different predicted target sequences, according to the presence or absence of Glu-9/Arg-12 residues in the basic region. Group (1A) had 180 putative E-boxbinding proteins with the conserved Glu-9/Arg-12 residues and group (1B) had 16 non-E-box-binding proteins lacking these residues. Three residues (His/Lys-5, Glu-9 and Arg-13) in the basic region characterize the classic G-box-binding proteins (Massari and Murre 2000). Thus, we could further subdivide group (1A) into two subgroups: 1A1 comprised G-box-binding proteins (145 members), and 1A2 comprised non-G-binding proteins (35 predicted members). Among the DNA-binding type bHLHs, the number of non-E-box binding proteins in the Chinese cabbage was fewer than that in rice (Supplementary Fig. 4).

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Mol Genet Genomics (2014) 289:77–91 35

BrabHLH112 BrabHLH204

30 BrabHLH225(-) BrabHLH168(-)

25 BrabHLH131

BrabHLH132

BrabHLH186 BrabHLH214(-)

20

BrabHLH154(-)

Mb

BrabHLH161

15

BrabHLH077 BrabHLH011(-)

BrabHLH219(-) BrabHLH212 BrabHLH104(-) BrabHLH106

BrabHLH226 BrabHLH211(-) BrabHLH103(-) BrabHLH105 BrabHLH135

BrabHLH134

BrabHLH133

BrabHLH009

10

BrabHLH049 BrabHLH215

BrabHLH216

BrabHLH191(-) BrabHLH136(-)

BrabHLH082 BrabHLH081 BrabHLH079(-) BrabHLH062(-) BrabHLH064

BrabHLH061(-) BrabHLH063(-) BrabHLH065 BrabHLH067(-) BrabHLH069

BrabHLH066(-) BrabHLH068

BrabHLH130(-)

BrabHLH197

BrabHLH140

BrabHLH129

BrabHLH167 BrabHLH041(-)

BrabHLH141(-)

BrabHLH005(-) BrabHLH003(-) BrabHLH001

BrabHLH183(-) BrabHLH173

BrabHLH174

BrabHLH152

BrabHLH139 BrabHLH166(-) BrabHLH040

BrabHLH039(-) BrabHLH038(-)

BrabHLH037(-)

A02

BrabHLH193

BrabHLH172

BrabHLH165(-)

BrabHLH164

A01

BrabHLH007

BrabHLH006 BrabHLH004(-) BrabHLH002

BrabHLH137

BrabHLH138(-)

BrabHLH080

40

BrabHLH169

BrabHLH075 BrabHLH076(-) BrabHLH078

BrabHLH008

BrabHLH050(-) BrabHLH048

0

BrabHLH121

BrabHLH218

BrabHLH170(-)

BrabHLH217(-)

5

BrabHLH120

BrabHLH010

BrabHLH162 BrabHLH177(-)

BrabHLH163(-)

BrabHLH142

BrabHLH181

BrabHLH182(-)

BrabHLH192

G-box binding Non-G-box binding Non-E-box binding Non-DNA binding

BrabHLH111

BrabHLH176(-) BrabHLH194(-)

BrabHLH227

BrabHLH175(-) BrabHLH205

BrabHLH117

BrabHLH116 BrabHLH114

BrabHLH115 BrabHLH036(-) BrabHLH089 BrabHLH087 BrabHLH085 BrabHLH083

A03

BrabHLH088 BrabHLH086 BrabHLH084

BrabHLH035(-) BrabHLH033 BrabHLH031 BrabHLH029(-)

BrabHLH034 BrabHLH032(-) BrabHLH030 BrabHLH028

A04

A05

BrabHLH184

35

BrabHLH178(-) BrabHLH180(-)

BrabHLH179 BrabHLH155(-)

BrabHLH224(-) BrabHLH222(-) BrabHLH046

BrabHLH223(-) BrabHLH047

30

BrabHLH045(-)

BrabHLH044(-) BrabHLH042(-)

BrabHLH043(-)

25

BrabHLH147(-)

Mb

BrabHLH150(-) BrabHLH210

BrabHLH053(-)

20

15

BrabHLH203(-)

BrabHLH190 BrabHLH146(-) BrabHLH148(-) BrabHLH151

BrabHLH149(-)

BrabHLH144

BrabHLH143

BrabHLH097 BrabHLH099(-) BrabHLH026 BrabHLH024 BrabHLH022(-)

BrabHLH096(-) BrabHLH098

BrabHLH102(-)

BrabHLH027 BrabHLH025 BrabHLH023(-)

BrabHLH100 BrabHLH059(-)

BrabHLH101 BrabHLH060

BrabHLH057(-) BrabHLH055

BrabHLH058 BrabHLH056(-) BrabHLH054(-)

BrabHLH195

BrabHLH196(-)

BrabHLH021

BrabHLH020(-) BrabHLH019(-) BrabHLH017(-)

BrabHLH018(-)

10

BrabHLH070 BrabHLH072

BrabHLH113(-)

BrabHLH153

BrabHLH122(-)

5

BrabHLH123(-) BrabHLH125 BrabHLH127(-)

BrabHLH124(-) BrabHLH126 BrabHLH128

BrabHLH118

BrabHLH119

0

A06

BrabHLH199(-)

BrabHLH145

BrabHLH160 BrabHLH108(-) BrabHLH110(-) BrabHLH156

BrabHLH051 BrabHLH185(-) BrabHLH159(-)

BrabHLH073 BrabHLH171(-)

BrabHLH090

BrabHLH013(-) BrabHLH202

BrabHLH157(-) BrabHLH201

BrabHLH093 BrabHLH091

BrabHLH092

BrabHLH012(-)

BrabHLH213

BrabHLH071(-)

BrabHLH074

BrabHLH052(-)

BrabHLH107(-) BrabHLH109(-)

BrabHLH206

BrabHLH220

A08

BrabHLH015

BrabHLH207

BrabHLH187

BrabHLH198(-)

BrabHLH189

BrabHLH014 BrabHLH016(-) BrabHLH188 BrabHLH094(-)

BrabHLH095

BrabHLH208(-) BrabHLH200

A07

BrabHLH158

BrabHLH209(-)

A09

A10

Fig. 2  Distribution of 230 bHLH genes on the ten Chinese cabbage chromosomes. The centromeric positions are shown according to the Chinese cabbage genome sequencing results. Four transcription factors on the scaffold (BrabHLH221, BrabHLH228, BrabHLH229 and BrabHLH230) could not be anchored onto a specific chromosome.

The scale is in megabases (Mb). The different colored lines of the bHLH genes on the chromosomes represent their DNA-binding activity pattern. The duplicated bHLH genes are connected with a yellow line (color figure online)

To further analyze the characteristic amino acid residues in the bHLH domains, we performed multiple sequence alignment of the Chinese cabbage, Arabidopsis and rice bHLH protein sequences (Supplementary Fig. 5; Supplementary Table 3). Nine conserved amino acids (Glu-9, Arg-10, Arg-12, Arg-13, Leu-25, Pro-30, Leu-51, Tyr-57 and Leu-61) were identified. Their identities were over 75 % in the consensus motif (Fig. 3). The Leu-25 residue in the helix region was the most conserved of all sites in

the bHLH domains. This indicated that this site might play important roles in promoting the formation of dimerization among bHLH proteins. This phenomenon was also shown by previous reports (Atchley et al. 1999). Generally, the distributions of conserved amino acids among the bHLH domains of both Chinese cabbage and Arabidopsis were very similar, with slight differences from that of the rice bHLHs, as expected from the evolutionary distances for bHLHs between Monocots and Eudicots plants.

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X 90%

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Fig. 3  Distribution of amino acids in the bHLH consensus motif among Chinese cabbage, Arabidopsis and rice. The numbers below the species name refer to the positions of the residues in the alignments of the studies

Phylogenetic analysis of the bHLH transcription factor family A phylogenetic tree was used to assess the evolutionary relationships among Chinese cabbage, Arabidopsis and rice, based on bHLH transcription factors (Fig. 4). We used the conserved bHLH transcription factor domains to construct the NJ phylogenetic tree with a bootstrap value of 1000. Twenty-six subfamilies were identified according to the clade support values, topology of the tree and the classification of the Arabidopsis and rice (Toledo-Ortiz et al. 2003; Li et al. 2006; Pires and Dolan 2010). There were no BrabHLH proteins in the XIII and XIV subfamilies; therefore, Chinese cabbage contained 24 bHLH subfamilies in our analyses. Furthermore, the conserved motifs for the bHLH subfamilies proteins were identified using the Meme program (Supplementary Fig. 10). We found that most bHLH proteins in the same subfamily had similar motifs, and Meme also provided the LOGOs (a visual representation of the frequency of a motif in a given position) of the protein motifs (Supplementary Fig. 6). In addition, most members in the same subfamilies had a similar intron/ exon structure. For example, members of subfamily VIIIb and III (d + e) had only one exon (Supplementary Fig. 7). This was further evidence to support the close evolutionary relationship in the same subfamily. These results were consistent with a previous report in rice and Arabidopsis (Li et al. 2006). Most of these subfamilies were consistent with the groups proposed by previous phylogenetic analyses of plant bHLH using Arabidopsis and rice sequences alone (Toledo-Ortiz et al. 2003; Li et al. 2006; Pires and Dolan 2010). We adopted the Arabidopsis bHLH group nomenclature proposed by Pires and Dolan (2010) to label

these subfamilies, with some modifications; for example, VIIIc(1) and VIIIc(2) were combined into VIIIc. The high degree of sequence divergence from other bHLH subfamilies meant that 14 bHLH proteins did not clearly fall into any of the 26 subfamilies and were classified as “Orphans”, which contained two BrabHLH proteins. The phylogenetic analyses of bHLH proteins have been reported for metazoans and for some plants and fungi (Ledent and Vervoort 2001; Simionato et al. 2007; Carretero-Paulet et al. 2010; Pires and Dolan 2010). They have provided a useful phylogenetic framework for the classification of bHLH proteins. Nevertheless, these analyses were solely based on the bHLH proteins of plants or metazoans. They provided no insight into the diversity of this family in the earlier diverging groups of plants and metazoans. To determine if these subfamilies were plant-specific or if they arose earlier in plant evolution and to understand the deeper evolutionary history of this family in plants, metazoans and fungi, we used all the collected, conserved bHLH regions to construct phylogenetic trees. Based on the topology of the trees, we defined 27 subfamilies of bHLH proteins in plants. The VI subfamily only contained the bHLH proteins of Selaginella moellendorffii and Physcomitrella patens (Supplementary Fig. 8a). In metazoans, the bHLH proteins mainly comprised six groups (groups A to F) (Supplementary Fig. 8b). Further analysis showed that six bHLH genes of fungi were gathered in group B, and several plant bHLH genes were also assigned to this group. In the six groups, only group B contained bHLH proteins of metazoans plants and fungi (Supplementary Fig. 8c, d). Thus, group B was likely to represent the ancestor of the bHLH proteins, which was consistent with a previous report (Atchley and Fitch 1997). However, some plant bHLH genes were found

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BrabHLH2 16 AthbHLH049 BrabHLH026 AthbHLH076 OsabHLH086 OsabHLH088 OsabHLH087 OsabHLH084 OsabHLH085 BrabH LH116 AthbH LH074 93 BrabHLH12 BrabHLH17 7 68 OsabHLH 9 176 BrabHLH BrabHLH 210 AthbHLH 131 AthbHL 078 BrabHL H062 OsabHL H169 83 OsabH H093 LH 079 OsabH BrabH LH083 AthbH LH139 98 OsabH LH137 55 Osab LH081 Osab HLH080 Brab HLH082 57 HLH0 Athb 66 HL H063 Brab 80 59 Brab HL H196 50 Brab HL H205 74 Brab HLH161 88 Athb HLH093 74 O sa HLH0 O sa bH LH0977 Osa bH LH09 1 bH LH 2 Br 81 ab 60 A th H LH 090 Osa bHLH 126 100 Osa bHLH 031 Bra bHLH 089 A th bHLH 094 Bra bHL 198 83 Bra bHL H079 99 Bra bHL H220 58 H bH Ath L 207 Bra bHL H144 Bra bHL H064 65 Ath bHL H05 Bra bH H11 5 98 A th bH LH05 2 Bra bH LH03 8 Bra bH LH0 4 Bra bH LH01 48 L b 9 A H H0 78 Bra thbH LH0 44 A bH LH 83 Bra thbH L H 060 73 A bH LH 060 A thbH LH 075 Bra thbH LH 153 B bH LH 04 O rabH L H 05 4 Osa sabH LH 049 0 B b L 09 B rab H L H0 7 B rab HL H0 96 B rab HL H1 97 A rab HL H2 17 A thb H L H1 00 O thb H L H0 89 Bra sab HL H0 07 bH HL H00 59 LH H09 7 01 8 3

XII

33 HLH0 51 Athb HL H1 6 Brab LH11 4 bH 70 A th H LH12 6 18 Brab H LH 2 Brab LH10 2 66 19 bH 68 Bra bHLH 002 Bra LH 1 00 bH 57 O sa bHLH 004 Osa bHLH 003 O sa bH LH 061 70 O sa bHLH 181 99 H A th bHL 093 0 10 Bra bHLH H143 80 Ath bHL H155 65 Bra bHL 005 97 H 2 Osa bHL LH01 70 O sa bH H021 Bra bH L H077 0 L Ath bH LH18 25 Bra bH H1 0 55 Bra bHL H09 29 Bra bHL LH0 93 96 1 Ath bH L H 070 Ath rabH LH 005 B bH LH 156 Bra bH LH 07 0 Bra bH LH 006 O sa sabH LH 027 2 O sabH LH 14 3 O thbH H LH H06 41 A rab H L H0 5 B rab HL 03 0 B b LH 13 4 Bra bH LH H01 4 6 th H A rab HL H1 22 B rab HL H0 81 B b HL H0 67 O sa thb HL H 1 A rab H L B sab O

IIIb

94

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B A rab B thb HL B rab HL H Ath rab HL H1 080 B b HL H0 60 O rab HLH H0 99 Bra sabH H LH 10 84 B b L 0 1 Bra rabH HL H H05 37 Bra bH L H 09 6 B bH LH 09 2 Bra rabH LH 043 1 B bH L H 22 A th rabH LH 042 9 L b Bra H H 018 Bra bH LH1 017 B bH LH 00 A th rabH LH 104 Ath bH L H0 032 L Bra bH L H03 88 63 Bra bHL H03 9 82 Bra bH H0 8 Osa bHL LH08 86 Osa bHL H08 7 55 O sa bHL H070 5 Osa bHL H06 Ath bHL H069 8 H bH 5 06 Ath 9 L 7 Ath bHLHH114 Bra bHLH 103 61 57 A th bHL 112 H Osa bHLH 177 80 Osa bHLH 111 Osa bHLH 077 99 Osa bHLH 076 A th bHLH 075 bH LH 071 A th bHLH 110 A th bH LH 113 O sa 88 bHLH 068 Osa 07 bH 82 82 Osab LH07 2 59 Osab HLH07 4 Athb HLH0783 HLH1 Osab 23 Athb HLH065 98 Athb HLH154 HLH1 Osab HLH0 53 OsabH 66 OsabH LH174 81 OsabH LH173 OsabH LH153 78 LH177 95 OsabH LH175 OsabH LH154 76 AthbH LH1 BrabHL 61 H11 97 BrabHLH 3 BrabHLH 141 051 AthbHLH 134 BrabHLH 99 AthbHLH 039 136 BrabHLH10 73 0 90 BrabHLH07 4 97 BrabH LH145 93 BrabHLH096 BrabHLH021 AthbHLH135 98 BrabHLH050 56 83 OsabHLH140 OsabHLH139 99 53 62 AthbHLH144 OsabHLH138 OsabHLH158 OsabHLH15 9 AthbHLH149 AthbHLH148 OsabHLH166 OsabHLH163 OsabHLH162 AthbHLH143

8 01 8 LH 07 2 bH H 17 sa HL H 24 O rab HL H2 020 B rab H L H 19 B rab HL H0 73 B thb HL H1 23 A thb HL H2 22 A rab HL H0 20 B rab HL H0 1 B b L 02 9 sa O sabH HLH H01 3 O sab H L 02 3 O sab HLH 21 2 O sab LH 04 O rabH LH 017 B thbH LH 157 A bH LH 158 90 Osa bH LH 002 BrarabH LH 152 95 H B H b 01 L 94 Ath bH LH0 12 87 Bra bH LH0 16 76 Ath bH LH 0 5 80 88 A th bH LH 01 3 Osa bH H01 4 O sa bH L H01 5 54 Osa bHL LH16 2 Osa bH H01 51 L 052 Osa bH H 97 O sa bHL H046 92 L 038 Bra bH Ath bHL HH168 Bra bHL 034 Bra bHLH 031 90 Osa bHLH 032 Osa bH LH 141 75 Osa bH LH 5 56 99 21 A th HLH 7 80 Brab HLH02 2 Brab LH1058 bH A th HL H0 5 67 Brab HLH03 36 Osab HLH0 94 Osab HLH037 62 Osab HLH042 99 90 Osab HLH043 75 Osab HLH031 Brab 105 LH 75 BrabHHLH106 Athb LH107 97 AthbH LH089 97 BrabH LH041 OsabH LH001 83 BrabH LH106 98 BrabH H051 AthbHL 8 82 H03 98 OsabHL H039 OsabHL H040 73 100 OsabHL 023 BrabHLH 030 AthbHLH 025 BrabHLH 32 AthbHLH0 0 BrabHLH15 BrabHLH154 AthbH LH108 BrabHLH191 AthbHLH109

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87

99 73

99 97 97

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9 12 0 LH H04 82 bH HL H0 14 Bra rab HL LH1 115 0 B thb H LH 10 A sab H LH 83 O sab H H1 66 O sab HL H0 47 O rab HL H0 4 B thb HL H06 9 A rab H L H06 2 B rab HL 20 0 B thb L H 08 0 95 A rabH H LH 16 1 B thb LH 08 A bH LH 194 Bra thbH LH 033 A bH L H 130 67 BrarabH LH 103 B thbH LH 112 A bH LH 43 61 BrasabH LH1 19 O bH LH1 2 73 9 Osa bH LH12 9 5 Bra bH LH10 1 62 Ath bH H21 0 69 Osa bHL H11 1 Bra bH L LH11 4 O sa bH H09 89 Osa bHL H128 8 Bra bH L H003 9 Ath bHL 035 59 Bra bHL H 129 65 Bra bH LH 065 79 Ath bHLH 195 Bra bHL H 204 Bra bHL H 085 Bra bHLH 134 A th bHLH 132 2 4 6 7 O sa bH LH 084 O sa bH LH 8 A th bH LH12 9 59 Osa HLH1231 Osab HLH1 Osab HL H171 Brab HLH139 Athb HLH130 Osab HLH059 85 Brab HLH1 Brab HLH054 Athb LH221 BrabH LH133 OsabH LH024 BrabH LH127 55 OsabH LH125 OsabH LH086 AthbH H126 OsabHL H122 77 BrabHL H083 AthbHL 036 BrabHLH 135 73 BrabHLH 140 57 85 52 68 BrabHLH 053 55 AthbHLH 83 2 AthbH LH05 8 99 OsabH LH17 96 BrabH LH165 94 AthbH LH140 79 OsabHLH123 OsabHLH122 91 BrabHLH176 96 AthbHLH087 71 BrabHLH010 OsabHLH117 OsabHLH118 OsabHLH119 100 Brab HLH00 8 82 AthbHLH040 51 BrabHLH164 AthbHLH043 54 OsabHLH124 90 OsabHLH120 OsabHLH121 BrabHLH203 BrabHLH206 77 BrabH LH071 BrabHLH170 AthbHLH08 BrabHLH 8 199 92 78 100 AthbHLH OsabHLH 037 OsabHL 116 OsabHL H136 H135 OsabHL AthbHL H137 AthbHL H023 AthbH H119 86 AthbH LH056 99 83 70 BrabH LH127 BrabH LH062 BrabH LH061 BrabH LH004 Athb LH028 100 HLH1 Brab 24 57 70 93 Athb HLH020 78 99 Brab HLH132 Brab HL H188 94 Athb HL H184 93 66 Brab HLH0 26 HL Athb H006 Brab HLH01 50 H 6 O sa LH20 100 Br ab bH LH 8 Ath H LH 113 Bra bH LH 076 66 Ath bHL H 072 Bra bHLH 068 76 O sa bHLH 024 89 O sa bH L 056 56 Bra bH L H 108 Ath bHL H107 Osa bHL H072 Ath bH L H073 53 Ath bHL H10 Bra bHL H00 1 Bra bHL H06 9 Bra bH H04 5 Ath bH L LH21 5 93 Osa bHL H00 2 O bH H0 2 99 O sabH LH1 15 O sasabH LH1 03 99 O bH LH 52 O sabH LH 102 6 A sabH LH 104 98 1 B thbH LH 106 74 Bra rabH LH 105 Ath bH LH 008 53 1 L A b 2 H 75 A thb HLH 17 8 O thb HL 11 8 O sab H L H15 7 O sab H L H1 5 59 B sab HL H1 56 A rab HL H1 49 B thb HL H 50 B rab HL H 151 B rab HL H 069 A rab HL H0 025 th H H 5 b H L H 22 7 LH 17 2 01 4 8

99

01 B 1 B rab HL H1 14 A rab H L H1 47 Ath thbH HL H1 46 Bra bH LH H10 08 99 Bra bH LH 02 7 B b L 00 8 Ath rab H LH H14 5 62 O sa bH HLH 10 8 71 A th bH LH 079 9 Bra bH LH 004 Osa bH LH 009 Ath bH LH 006 0 L Bra bH H0 53 B bH LH0 10 54 Osa rabH LH2 03 Ath bHL LH0 17 Bra bHL H00 75 82 64 Bra bHL H01 8 Bra bHL H187 3 93 A th bHL H030 Osa bH L H21 57 63 Osa bHL H017 9 98 Ath bHL H142 H Bra bHLH 141 58 Bra bHL H 091 A th bHL 133 Ath bH LH H114 bH Bra LH 138 93 Bra bHLH 089 Ath bHL H 095 99 bH 11 Brab LH01 5 98 Osa HLH 0 bH LH 134 Osa bHLH 027 O sa 02 Osa bH LH02 5 70 bH Athb LH02 6 HLH0 8 69 Brab 7 41 0 95 Osab HLH197 72 98 61 56 Osab HLH024 59 HLH0 Osab HLH0 30 Brab HLH1 29 99 Brab HLH1 90 Athb 56 56 HL OsabH H092 91 OsabH LH171 100 OsabH LH148 92 OsabH LH146 66 LH BrabHL 144 95 AthbHL H227 BrabHL H095 AthbHL H118 H099 BrabHLH 99 10 182 0 77 AthbHLH 90 74 BrabHLH098 BrabHLH 137 71 167 BrabHLH01 97 OsabH LH05 6 4 79 OsabH LH05 3 OsabH LH055 72 BrabHLH009 AthbHLH045 BrabHLH225 BrabHLH132 80 70 74 98 AthbHLH070 BrabHLH226 74 85 52 BrabHLH029 OsabHLH052 AthbHLH0 67 64 BrabHLH046 97 BrabHLH149 50 BrabHLH110 AthbHLH071 AthbHLH057 BrabHLH209 96 86 99 58 163 BrabHLH BrabHLH011 AthbH LH097 OsabH LH051 54 2 BrabHLH16 50 OsabHLH0 049 OsabHLH 159 BrabHLH 048 OsabHLH 046 OsabHLH 2 H02 BrabHL H048 BrabHL H096 AthbHL H098 bHL Bra LH045 60 OsabH LH047 bH Osa LH044 100 OsabH LH094 89 AthbH LH228 95 BrabH LH101 52 BrabH H073 HL Brab H121 HL Athb HL H214 86 Brab HLH064 69 66 Osab HL H111 88 79 Brab H011 7 HL A thb HLH06 7 Brab LH04 1 77 bH Ath bH LH06 3 06 O sa bHLH 062 91 Osa 66 bHLH 059 Osa bHLH 058 67 O sa bHLH 057 Osa bHLH 060 98 Osa bHLH 104 99 60 Osa bH LHH230 A th bHL 034 71 Bra bH LH 090 H Ath bHL H115 61 Bra bHL H175 1 5 6 9 0 8 Ath bHL H16 8 10 Bra bHL H13 88 Bra bHL H105 Bra bHL H0157 74 Ath bHL H14 0 Bra bHL H17 55 97 L Osa bH LH1 23 96 51 Osa bH LH1 55 Bra bH LH0 25 Bra bH H1 01 L 2 Ath bH LH 20 1 Ath rabH LH 054 B bH LH 120 83 3 0 A th rabH LH 126 2 8 B bH LH 08 1 10 Bra bH LH 12 Ath rabH H LH 118 6 B rab LH 03 6 B bH LH 13 9 88 H 6 Ath thbH HL H1 68 A rab H L H1 72 B sab HL H1 62 O sab HL H1 18 O sab HL H2 O thb bHL A ra B

68

III(d+e)

O A sab B thb HL Bra rabH HLH H b L 0

53

Orphans

56

III(a+c)

XIV Orphans

IIIf

Va

Vb

Fig. 4  Phylogenetic tree constructed from the neighbor-joining method using bHLH transcription factor domains in Chinese cabbage, Arabidopsis and rice. The phylogenetic tree was constructed using

MEGA5. The numbers are bootstrap values based on 1,000 iterations. Only bootstrap values larger than 50 % support are indicated

in group D, which may provide a new perspective for investigating the origin and evolution of bHLH proteins.

but with the addition of a WGT, thought to have occurred between 13 and 17 million years ago (mya) (Wang et al. 2011). Extensive analyses of the complete genome sequence of rice support one ancient WGD event (σ) that occurred early in the monocot lineage. Two WGD events (ρ and σ) have been inferred that pre-date the diversification of cereal grains and other grasses (Poaceae) (Tang et al. 2010; Jiao et al. 2011). Comparative genomic analysis confirmed that Chinese cabbage underwent genome triplication since its divergence from Arabidopsis. Therefore, many collinear blocks were observed between the Chinese cabbage and Arabidopsis genomes. Interestingly, the gene number in the Chinese

Orthologous and paralogous bHLH genes in Chinese cabbage, Arabidopsis and rice Most Angiosperm plant lineages have experienced one or more rounds of ancient polyploidy (Lee et al. 2013). For example, Arabidopsis has undergone two recent whole genome duplications (WGD: α and β) within the Brassicaceae lineage, and one whole genome triplication event (WGT: γ) that may be shared by all core eudicots. B. rapa shares this complex evolutionary history with Arabidopsis,

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Fig. 5  Comparative analysis of synteny and expansion of bHLH genes. a Ten Chinese cabbage (A01–A10) and five Arabidopsis chromosome (Chr1–Chr5) maps were based on the orthologous and paralogous pair positions and demonstrate highly conserved synteny. The red lines represent the orthologous bHLH genes between Chinese cabbage and Arabidopsis. The blue and green lines represent the paralogous bHLH genes in Chinese cabbage and Arabidopsis,

respectively. b Ten Chinese cabbage (A01–A10) and rice chromosome (Chr1–Chr12) maps were based on the orthologous and paralogous pair positions and demonstrate highly conserved synteny. The purple lines represent the orthologous bHLH genes between Chinese cabbage and rice. The blue and yellow lines represent the paralogous bHLH genes in Chinese cabbage and rice, respectively (color figure online)

cabbage genome was notably less than three times that of the Arabidopsis gene number. These results indicated gene loss during polyploid speciation. Comparative analysis then was used to identify orthologous bHLH transcription factors to assess bHLH gene triplication between Chinese cabbage and Arabidopsis. Using OrthoMcl, we identified 124 orthologous gene pairs among all the bHLH proteins between Chinese cabbage and Arabidopsis. Only 37 bHLH orthologous gene pairs were found between Chinese cabbage and rice, and 40 orthologous gene pairs between Arabidopsis and rice (Supplementary Table 4), which was consistent with the close relationship of the Chinese cabbage and Arabidopsis. Among the orthologous gene pairs between Chinese cabbage and Arabidopsis, we found that each Arabidopsis bHLH gene had one to three Chinese cabbage orthologous genes. These results demonstrated that in Chinese cabbage, a few bHLH transcription factor genes were duplicated accompanied by genome triplication. In addition, 59, eight and 69 paralogous bHLH gene pairs were identified in Chinese cabbage, Arabidopsis and rice, respectively (Supplementary Table 5). The Circos program was used to display the relationships of orthologous and paralogous bHLH genes among these three species (Fig. 5).

To gain further insight into the correlations among bHLH genes in Chinese cabbage, the interaction network of bHLH genes in Chinese cabbage was constructed using these orthologous transcription factors with Arabidopsis (Fig. 6). Pearson correlation coefficients of 153 gene pairs were greater than zero, and 28 gene pairs were less than zero. The orthologous and paralogous genes are listed in Supplementary Table 6. Chromosome distribution of the bHLH transcription factor family Among the 230 bHLH transcription factors, 226 were mapped onto the ten Chinese cabbage chromosomes, and the other four transcription factors were anchored in the scaffolds (BrabHLH221, BrabHLH228, BrabHLH229 and BrabHLH230; accession numbers Bra039819, Bra040856, Bra041013 and Bra041033, respectively, in the Chinese cabbage databank) (Fig. 2; Supplementary Table 2). Most of the bHLH transcription factors were found on chromosomes 9 (35, 15.2 %) and 3 (31, 13.5 %). In contrast, there were only 12 (5.2 %) and 13 (5.7 %) bHLH transcription factor genes on chromosomes 10 and 8. All the bHLH family transcription factors

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PCC >0 PCC =0 Bra022104

PCC 90 %) (Fig. 2). Interestingly, this may have been affected by the cabbage genome triplication. Fifteen of these duplicated genes were divided into five groups (BrabHLH225/132/009, BrabHLH219/030/053, BrabHLH050/096/021, BrabHLH141/039/051 and BrabHLH074/100/145) with exactly three bHLH genes with high sequence similarity in each group. These potentially triplicated genes were distributed on the ten chromosomes. The number of triplicated genes on chromosome 2 (four) was more than on the other nine chromosomes. Expression analysis of transcription factor family genes under different abiotic stresses Several bHLH transcription factors are important for plant growth in metabolic, biosynthesis and regulatory pathways, especially under abiotic stress (Zuo et al. 2004). Based on the qRT-PCR, EST database, MPSS and microarray data, previous analyses showed that 113 and 134 bHLH genes had detected expression signals in Arabidopsis and rice, respectively. This suggested that many bHLHs play regulatory roles in rice and Arabidopsis. The expression pattern of a gene is often correlated with its function; therefore, we carefully selected 93 representative BrabHLH genes and performed comprehensive expression analysis using qRTPCR. We analyzed the expression of the BrabHLHs using qRT-PCR with RNA extracted from the leaves under different abiotic treatments. The results of the qRT-PCR experiment provided a preliminary impression of the expression profiles of these genes. The relative expression level of BrabHLHs for each treatment was calculated with respect to the controls. The majority of genes were upregulated under PEG and cold treatment, while most genes were downregulated under ABA, GA and heat treatment (Fig. 7; Supplementary Table 7).

87

Until now, only a few Brassica rapa bHLH genes have been reported. For example, BrTT8 (BrabHLH213) regulates seed coat color (Li et al. 2012) and BrICE1 (EU 374158) is involved in the cold response (Jiang et al. 2011), Several genes are regulated by dehydration treatments, such as BRU03474 and BRU11590, which were downregulated, and BRU08596 and BRU01634, which were upregulated under drought conditions (Yu et al. 2012). Although the function of most BrabHLH genes is unknown, the phylogenetic and expression analyses provide a solid foundation for future functional studies. Identification of putative orthologs in different species will benefit the study of BrabHLHs’ functions, such as BrabHLH102, BrabHLH151, BrabHLH186, BrabHLH124, BrabHLH192, BrabHLH181 and BrabHLH143. They had high sequence similarity with the AtbHLH033 and AtbHLH116, which are involved in cold acclimation and freezing tolerance response. Thus, we deduced that these BrabHLHs might also be regulated by cold treatment. This hypothesis was proved using qRTPCR. In particular, BrabHLH143 was upregulated after 12 h of cold treatment (Fig. 8a). Three BrabHLHs (BrabHLH212, BrabHLH045 and BrabHLH002) were homologous with PIF4 (AtbHLH009) of Arabidopsis, which was shown to mediate plant architecture responses to high temperatures. In our analysis, BrabHLH212 and BrabHLH045 were downregulated after heat treatment, while the BrabHLH002 was initially up- and then downregulated (Fig. 8c). Furthermore, we also analyzed the expression of several ABA and GA response genes in Chinese cabbage (Fig. 8b, d), which showed that most orthologous genes in different species had similar expression patterns. Under abiotic stress treatments, although several BrabHLH genes have relatively low expression levels, the 93 BrabHLH genes were expressed in one or more of the conditions. Under cold, ABA, PEG and GA treatments, the relative expression levels of 28 BrabHLH members were five times higher than the control (Supplementary Fig. 9). Under heat stress, the relative expression values of BrabHLH members were not larger than 5. The expressions of five BrabHLH members (BrabHLH148, BrabHLH147, BrabHLH112, BrabHLH055, BrabHLH125) were over 20 times higher than the control after 12 h of cold stress. Under ABA treatment, BrabHLH045 and BrabHLH002 were upregulated at 4 h and then downregulated at 12 h. Under PEG stress, the relative expression levels of four BrabHLH members (BrabHLH212, BrabHLH116, BrabHLH143, BrabHLH061) exceeded 20 after 4 h and then decreased rapidly to an expression level lower than 10 at 12 h. Interestingly, only the expression value of BrabHLH148 was larger than 10 after 12 h under PEG stress. Furthermore, its expression value was higher than 70 after 12 h under cold stress. This indicated that BrabHLH148 might play an important positive regulatory role in cold

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BrabHLH131 BrabHLH125 BrabHLH195 BrabHLH158 BrabHLH213 BrabHLH152 BrabHLH066 BrabHLH191 BrabHLH007 BrabHLH180 BrabHLH099 BrabHLH072 BrabHLH174 BrabHLH055 BrabHLH147 BrabHLH056 BrabHLH148 BrabHLH043 BrabHLH184 BrabHLH068 BrabHLH012 BrabHLH124 BrabHLH230 BrabHLH151 BrabHLH134 BrabHLH179 BrabHLH127 BrabHLH189 BrabHLH194 BrabHLH181 BrabHLH090 BrabHLH028 BrabHLH062 BrabHLH022 BrabHLH061 BrabHLH101 BrabHLH208 BrabHLH192 BrabHLH222 BrabHLH079 BrabHLH018 BrabHLH098 BrabHLH085 BrabHLH110 BrabHLH169 BrabHLH006 BrabHLH093 BrabHLH105 BrabHLH076 BrabHLH229 BrabHLH073 BrabHLH121 BrabHLH088 BrabHLH002 BrabHLH067 BrabHLH115 BrabHLH206 BrabHLH156 BrabHLH039 BrabHLH086 BrabHLH038 BrabHLH020 BrabHLH004 BrabHLH081 BrabHLH116 BrabHLH065 BrabHLH199 BrabHLH197 BrabHLH210 BrabHLH202 BrabHLH112 BrabHLH190 BrabHLH188 BrabHLH157 BrabHLH077 BrabHLH228 BrabHLH036 BrabHLH045 BrabHLH141 BrabHLH128 BrabHLH143 BrabHLH196 BrabHLH212 BrabHLH111 BrabHLH031 BrabHLH186 BrabHLH177 BrabHLH102 BrabHLH178 BrabHLH052 BrabHLH135 BrabHLH048 BrabHLH205

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Fig. 7  Heat map representation and hierarchical clustering of BrabHLH genes during ABA, GA, heat, cold and PEG treatments. These expression profile data were obtained using qRT-PCR. The relative expression values were log2 transformed, and the heat map was generated using cluster software

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Fig. 8  The relative expression levels of several candidate bHLH genes during a cold, b ABA, c heat and d GA treatments. Error bars represent standard errors from three independent replicates

and drought stress. Under GA treatment, only one member (BrabHLH134) had an expression level higher than 10 at 12 h. The analyses of expression patterns and functional characterization of the BrabHLHs identified in this study may provide the basis for further study of the functions of these bHLH proteins in Chinese cabbage.

Discussion Chinese cabbage is a member of the Brassica genus and is one of the most important vegetables cultivated worldwide. Arabidopsis, a dicotyledonous species, was the first taxon to have its whole genome sequenced and released. Subsequently, more and more species genomes have been sequenced, such as the dicot species potato and tomato and the monocot species rice. In addition, many metazoans’ genomes have been sequenced. These data provide us with rich resources for comparative genomic analyses. Furthermore, with the rapid development in bioinformatics analyses, the information stored in various genomes may be explored to elucidate the mechanisms regulating the growth

and development of species. Comparative genomic analyses could also reveal the genome and gene evolution among plants, metazoans and fungi. In the present study, we analyzed the bHLH transcription factor family in Chinese cabbage and 37 other species, including 24 plants, 12 metazoans and one fungus. A total of 3,948 bHLH transcription factors were identified and analyzed in our research. The density of bHLH transcription factor genes in the Chinese cabbage genome was only less than that of Arabidopsis among all species analyzed. Compared with the species analyzed in this study, the Chinese cabbage genome supports a relatively large bHLH family. The greatest number of bHLH transcription factors was identified in Zea mays and Glycine max. In lower plants (i.e. Ostreoscoccus tauri and Cyanidioschyzon merolae), only one transcription factor is present because of their relatively small genomes. In metazoans, the number of bHLH transcription factors in Homo sapiens was more than in the other metazoans. In the analyses, the number of bHLH transcription factors in all higher plant and metazoans was more than that in the lower plants and fungi. In particular, the number of bHLH proteins increased

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with plant evolution and genome duplication. This suggested that the bHLH proteins might play very important roles in a species’ evolution. Comparative and phylogenetic analyses in Chinese cabbage and other species served as the first step in a comprehensive functional characterization of bHLH transcription factors using reverse genetic approaches and molecular genetics research. Recent research in functional and structural genomics in higher plant model species has shown that the bHLH transcription factors are involved in stress responses and plant development, including cold (AtbHLH116 and AtbHLH003), heat (AtbHLH009), abscisic acid, jasmonic acid and light signaling pathways (AtbHLH006) (Pires and Dolan 2010). Furthermore, bHLH transcription regulators are involved in plant metabolite biosynthesis and trait development, e.g. formation of root hairs (AtbHLH083), anther development (AtbHLH021) and axillary meristem generation (OsbHLH123) (Pires and Dolan 2010). However, there are very few reports about the bHLH genes in Chinese cabbage until now. Therefore, it was thought essential to identify and annotate the bHLH genes of Chinese cabbage. In the present study, we identified all the bHLH transcription factors in the whole genome of Chinese cabbage and characterized their expression patterns under different stress treatments. Interestingly, BrabHLH148 and BrabHLH212 were expressed highly under cold (12 h) and PEG (4 h) treatments, respectively. Their relative expressions exceeded 45 times that of the control. A comparison of species homologs, including protein sequences and expression profiles, might aid the understanding of the role of these transcription factors in Chinese cabbage. Generally, transcription regulators within the same taxonomic group may exhibit recent common evolutionary origins and specific conserved motifs related to molecular functions. It is an effective and practical way to predict unknown protein functions. The close relationship between Chinese cabbage and Arabidopsis permitted highly homologous genes to be identified and used to predict their functions in Chinese cabbage. Finally, we identified three bHLH genes that showed high similarity (>95 %) with the corresponding genes in Arabidopsis. Following the homologous gene annotations in Arabidopsis, we determined the functions of three BrabHLH genes in Chinese cabbage. For example, the BrabHLH039 (Bra006286) transcription factor exhibited high sequence similarity with the AthbHLH134 (At5g15160) gene. The Arabidopsis gene product is directly and negatively regulated by AP3 and PI in petals and is required for the appropriate regulation of flowering time (Mara et al. 2010). Both BrabHLH021 (Bra003797) and BrabHLH050 (Bra008154) transcription factors showed high sequence similarity with AthbHLH135 (At1g74500), which mainly acts as a positive regulator of gibberellin signaling and regulates AIF1 positively in

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response to the BR signaling pathway (Lee et al. 2006; Wang et al. 2009a). In summary, 230 bHLH transcription factors were identified in the whole Chinese cabbage genome. The structure, classification, evolutionary and expression patterns of this gene family were analyzed. Isolation and identification of these transcription factors are likely to assist in clarifying the molecular genetics basis for Chinese cabbage genetic improvement and also provide functional gene resources for transgenic research. The expression analysis may provide a solid foundation for future studies of bHLH protein regulatory functions during growth and development of Chinese cabbage. These data are also useful for constructing the protein interaction networks that control the growth of Chinese cabbage. Therefore, our results will pave the way for studies of the functions of bHLH genes in Chinese cabbage and will further our understanding of this gene family in other plants. In addition, the comparative genomic analysis of the bHLH genes has provided comprehensive insights into the evolutionary diversification of plants, metazoans and fungi. The expansion of the bHLH genes in Chinese cabbage was attributed to duplications during evolution, which showed that this gene family might play important roles in the polyploid crop. In conclusion, this is the first comprehensive and systematic analysis of bHLH transcription factors in Chinese cabbage. The results of this study revealed the importance of bHLH genes during growth and development. It may assist in elucidating bHLH family gene function in protein interactions, signaling pathway regulations and defense responses under different stress conditions. The bioinformatics analysis results provided basic resources for studying bHLH protein function for improving economic, agronomic and ecological benefit in Chinese cabbage and other species. Acknowledgments  This work was supported by the National High Technology Research and Development Program of China (863 Program, No. 2012AA100101), China Agriculture Research System CARS-25-A-12, the Fundamental Research Funds for the Central Universities of China (KYZ201111), and a Project Funded by the Priority Academic Program Development of Jiansu Higher Education Institutions.

References Atchley WR, Fitch WM (1997) A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA 94:5172–5176 Atchley WR, Terhalle W, Dress A (1999) Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J Mol Evol 48:501–516 Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS (2009) MEME SUITE: tools for motif discovery and searching. Nucl Acids Res 37:W202–W208

Mol Genet Genomics (2014) 289:77–91 Beilstein MA, Nagalingum NS, Clements MD, Manchester SR, Mathews S (2010) Dated molecular phylogenies indicate a Miocene origin for Arabidopsis thaliana. Proc Natl Acad Sci USA 107:18724–18728 Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martinez-Garcia JF, Bilbao-Castro JR, Robertson DL (2010) Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiol 153:1398–1412 Dang C, Wang Y, Zhang D, Yao Q, Chen K (2011) A genome-wide survey on basic helix-loop-helix transcription factors in giant panda. PLoS ONE 6:e26878 Douzery EJ, Snell EA, Bapteste E, Delsuc F, Philippe H (2004) The timing of eukaryotic evolution: does a relaxed molecular clock reconcile proteins and fossils? Proc Natl Acad Sci USA 101:15386–15391 Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20:735–747 Jiang FL, Wang F, Wu Z, Li Y, Shi GJ, Hu JD, Hou XL (2011) Components of the Arabidopsis CBF Cold-Response Pathway Are Conserved in Non-heading Chinese Cabbage. Plant Mol Biol Rep 29:525–532 Jiao Y, Wickett NJ, Ayyampalayam S, Chanderbali AS, Landherr L, Ralph PE, Tomsho LP, Hu Y, Liang H, Soltis PS, Soltis DE, Clifton SW, Schlarbaum SE, Schuster SC, Ma H, Leebens-Mack J, dePamphilis CW (2011) Ancestral polyploidy in seed plants and angiosperms. Nature 473:97–100 Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645 Ledent V, Vervoort M (2001) The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis. Genome Res 11:754–770 Ledent V, Paquet O, Vervoort M (2002) Phylogenetic analysis of the human basic helix-loop-helix proteins. Genome Biol 3: RESEARCH0030 Lee S, Yang KY, Kim YM, Park SY, Kim SY, Soh MS (2006) Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana. Plant Cell Physiol 47:591–600 Lee TH, Tang HB, Wang XY, Paterson AH (2013) PGDD: a database of gene and genome duplication in plants. Nucl Acids Res 41:D1152–D1158 Li L, Stoeckert CJ Jr, Roos DS (2003) OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13:2178–2189 Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z, Guo J, Liang W, Chen L, Yin J, Ma H, Wang J, Zhang D (2006) Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol 141:1167–1184 Li X, Chen L, Hong M, Zhang Y, Zu F, Wen J, Yi B, Ma C, Shen J, Tu J, Fu T (2012) A large insertion in bHLH transcription factor BrTT8 resulting in yellow seed coat in Brassica rapa. PLoS ONE 7:e44145 Liu WY, Zhao CJ (2010) Genome-wide identification and analysis of the chicken basic helix-loop-helix factors. Comp Funct Genomics 2010:1–12 Mara CD, Huang T, Irish VF (2010) The Arabidopsis floral homeotic proteins APETALA3 and PISTILLATA negatively regulate the BANQUO genes implicated in light signaling. Plant Cell 22:690–702 Massari ME, Murre C (2000) Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol Cell Biol 20:429–440

91 Murre C, McCaw PS, Baltimore D (1989) A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56:777–783 Peterson KJ, Lyons JB, Nowak KS, Takacs CM, Wargo MJ, McPeek MA (2004) Estimating metazoan divergence times with a molecular clock. Proc Natl Acad Sci USA 101:6536–6541 Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29:e45 Pires N, Dolan L (2010) Origin and diversification of basic-helixloop-helix proteins in plants. Mol Biol Evol 27:862–874 Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer EL, Eddy SR, Bateman A, Finn RD (2012) The Pfam protein families database. Nucl Acids Res 40:D290–D301 Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504 Simionato E, Ledent V, Richards G, Thomas-Chollier M, Kerner P, Coornaert D, Degnan BM, Vervoort M (2007) Origin and diversification of the basic helix-loop-helix gene family in metazoans: insights from comparative genomics. BMC Evol Biol 7:33 Skinner MK, Rawls A, Wilson-Rawls J, Roalson EH (2010) Basic helix-loop-helix transcription factor gene family phylogenetics and nomenclature. Differentiation 80:1–8 Stevens JD, Roalson EH, Skinner MK (2008) Phylogenetic and expression analysis of the basic helix-loop-helix transcription factor gene family: genomic approach to cellular differentiation. Differentiation 76:1006–1022 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tang H, Bowers JE, Wang X, Paterson AH (2010) Angiosperm genome comparisons reveal early polyploidy in the monocot lineage. Proc Natl Acad Sci USA 107:472–477 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882 Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helixloop-helix transcription factor family. Plant Cell 15:1749–1770 Wang H, Zhu Y, Fujioka S, Asami T, Li J (2009a) Regulation of Arabidopsis brassinosteroid signaling by atypical basic helixloop-helix proteins. Plant Cell 21:3781–3791 Wang Y, Chen K, Yao Q, Zheng X, Yang Z (2009b) Phylogenetic analysis of zebrafish basic helix-loop-helix transcription factors. J Mol Evol 68:629–640 Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, Cheng F (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039 Yu SC, Zhang FL, Yu YJ, Zhang DS, Zhao XY, Wang WH (2012) Transcriptome profiling of dehydration stress in the Chinese cabbage (Brassica rapa L. ssp pekinensis) by tag sequencing. Plant Mol Biol Rep 30:17–28 Zhang H, Jin J, Tang L, Zhao Y, Gu X, Gao G, Luo J (2011) PlantTFDB 2.0: update and improvement of the comprehensive plant transcription factor database. Nucl Acids Res 39:D1114–D1117 Zheng X, Wang Y, Yao Q, Yang Z, Chen K (2009) A genome-wide survey on basic helix-loop-helix transcription factors in rat and mouse. Mamm Genome 20:236–246 Zuo K, Zhao J, Wang J, Sun X, Tang K (2004) Molecular cloning and characterization of GhlecRK, a novel kinase gene with lectin-like domain from Gossypium hirsutum. DNA Seq 15:58–65

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