Mol Biol Rep DOI 10.1007/s11033-013-2980-x

Identification and characterization of the SET domain gene family in maize Yexiong Qian • Yilong Xi • Beijiu Cheng Suwen Zhu • Xianzhao Kan

•

Received: 2 November 2012 / Accepted: 24 December 2013 Ó Springer Science+Business Media Dordrecht 2014

Abstract Histone lysine methylation plays a pivotal role in a variety of developmental and physiological processes through modifying chromatin structure and thereby regulating eukaryotic gene transcription. The SET domain proteins represent putative candidates for lysine methyltransferases containing the evolutionarily-conserved SET domain, and important epigenetic regulators present in eukaryotes. In recent years, increasing evidence reveals that SET domain proteins are encoded by a large multigene family in plants and investigation of the SET domain gene family will serve to elucidate the epigenetic mechanism diversity in plants. Although the SET domain gene family has been thoroughly characterized in multiple plant species including two model plant systems, Arabidopsis and rice, through their sequenced genomes, analysis of the entire SET domain gene family in maize was not completed following maize (B73) genome sequencing project. Here, we performed a genome-wide structural and evolutionary analysis of maize SET domain genes from the latest

Y. Qian (&)  Y. Xi  X. Kan Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, China e-mail: [email protected]

version of the maize (B73) genome. A complete set of 43 SET domain genes (Zmset1-43) were identified in the maize genome using Blast search tools and categorized into seven classes (Class I–VII) based on phylogeny. Chromosomal location of these genes revealed that they are unevenly distributed on all ten chromosomes with seven segmental duplication events, suggesting that segmental duplication played a key role in expansion of the maize SET domain gene family. EST expression data mining revealed that these newly identified genes had temporal and spatial expression pattern and suggested that many maize SET domain genes play functional developmental roles in multiple tissues. Furthermore, the transcripts of the 18 genes (the Class V subfamily) were detected in the leaves by two different abiotic stress treatments using semiquantitative RT-PCR. The data demonstrated that these genes exhibited different expression levels in stress treatments. Overall, our study will serve to better understand the complexity of the maize SET domain gene family and also be beneficial for future experimental research to further unravel the mechanisms of epigenetic regulation in plants. Keywords Chromatin  Histone  Lysine methylation  SET domain  Zea mays L.

Y. Xi e-mail: [email protected] X. Kan e-mail: [email protected] B. Cheng  S. Zhu Key Laboratory of Crop Biology, Anhui Agricultural University, Hefei 230036, China e-mail: [email protected] S. Zhu e-mail: [email protected]

Introduction In eukaryotes, multiple epigenetic mechanisms are involved in regulating the structure and function of chromatin, which is a key regulator that influences the accessibility of factors and cofactors for all DNA-templated processes [1]. As a fundamental unit of chromatin, the nucleosome is composed of *146 base pairs of DNA

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around a histone octamer (containing H3–H4 tetramer and two H2A–H2B dimers) [2]. In nucleosomal structure, specific chemical and posttranslational modifications of these histones have been elucidated to play vital roles in eukaryotic gene regulation [3–5]. Covalent modifications of the N-terminal histone tails include methylation, acetylation, phosphorylation, glycosylation, ADP-ribosylation, sumoylation and ubiquitination [6–8] ,which are called the basis of the histone code that regulate gene expression epigenetically through various mechanisms [6, 9–11]. Among them, histone methylation is one of the most complex modifications, which not only occurs at different residues (lysine and arginine) and distinct sites but also differs in the number of methyl groups added [1]. At the moment, there are at least six lysine residues on histone H3 (K4, K9, K27, K36, K79) and H4 (K20) that are targeted by histone lysine methyltransferases (HKMTs) [1, 2]. The histone methylation at these specific lysine residues can participate in multiple developmental processes including cell cycle regulation, heterochromatin formation, stress responses, transcriptional activation and silencing in many organisms [12, 13]. In addition to the specific lysine residue, H3K79, catalyzed by a unique histone lysine methyltransferase DOT1 [14], most of histone lysine methylation residues are regulated by SET (Su(var)3–9, E(Z) and Trithorax) domain proteins, which are putative candidates for HKMTs [1, 13]. In plants, the SET domain-containing proteins commonly belong to an evolutionarily conserved and large multigene family. For instance, in recent years, reports of genomewide structural and evolutionary analysis of the entire SET domain gene family have been revealed in two model plant systems, Arabidopsis and rice [15]. The Arabidopsis and rice genomes encode at least 47 and 43 SET domain proteins, respectively [2, 16]. Of them, Arabidopsis SET domain proteins are the best annotated and characterized. According to the most recent report, Arabidopsis SET domain proteins can be classified into at least seven distinct classes with acting on different lysine residue for each one: (1) Enhancer of zeste [E(Z)] homologs (H3K27); (2) The ASH1 homologs (H3K36); (3) The trithorax homologs and related proteins (H3K4); (4) Proteins with a SET and a PHD domain; (5) Suppressor of variegation [Su(var)] homologs and relatives (H3K9); (6) Proteins with an interrupted SET domain; and (7) RBCMT and other SETrelated proteins. It has been demonstrated that diverse mechanisms could be involved in shaping the function and regulation of these SET domain proteins [2]. Although the enzymatic activity and specificity of these plant SET domain proteins remain unclear in every case, increasing experimental data revealed that they may act on the same lysine residues or related pathways to the homologous proteins or protein complexes in animals or

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yeast [1]. Furthermore, their activity, including changing degrees of methylation (mono-, di- or tri-methylation), substrate specificity and localization can be subjected to regulation by intra- and inter-molecular interactions with other proteins, which may be important in plant developmental processes, such as flowering time control and embryogenesis [2, 13]. Arabidopsis genome encodes three E(Z) homologs, CURLY LEAF (CLF), MEDEA (MEA), and SWINGER (SWN) that are widely believed to be H3K27me3 methyltransferases. CLF, the first identified plant SET domain-containing protein within this class [17], has been suggested to regulate flowering time as well as leaf and flower morphology through stable repression of the floral homeotic genes AGAMOUS (AG) and SHOOTMERISTEMLESS (STM) [18, 19]. MEA plays a role in maintaining its own imprinting during endosperm development and methylation of H3K27 is associated with MEA silencing during vegetative development and male gametogenesis [20–22]. SWN exhibits partial functional redundancy with CLF and MEA: swn mutants have no obvious phenotypes but strongly enhance the phenotypes of clf and mea mutants in clf swn and mea swn double mutants [23–25]. There are at least four ASH1 homologs and three ASH1-related proteins in Arabidopsis genome [26]. Among them, the Arabidopsis ASH1 HOMOLOG proteins, ASHH1 and ASHH2, have been suggested to exhibit H3K36 methyltransferase activity and are similar to yeast Set2, a H3K36 HMTase. Moreover, Mutation of the ASHH2 gene has been revealed that an early flowering phenotype results from a decrease in H3K36 methylation at the FLOWERING LOCUS C (FLC) [27]. The first Arabidopsis trithorax homolog, ATX1 has been confirmed to methylate H3K4 and regulates floral development through activating flower homeotic genes in plants [28, 29]. ATXR5 and ATXR6 were originally classified as TRX related, but now constitute a class of their own (Class IV) with a SET and a PHD domain [2, 16, 30]. Recent data suggested that ATXR5 and ATXR6 exhibits H3K27 monomethyltransferase activity and mutants show partial heterochromatin decondensation [31]. In Arabidopsis, the Su(var) homologs and relatives, SUVH and SUVR proteins, mainly regulate heterochromatin formation by achieving various functional H3K9 methylation states that will eventually lead to DNA methylation in a locus-specific manner [32]. Although the SET domain gene family has been thoroughly characterized in Arabidopsis, rice and other species through their sequenced genomes [13, 15, 33], many aspects of the role that SET domain proteins may play in plant development and the detailed mechanism by which they regulate chromatin structure and gene activity remain unclear. Especially, very little is known about the functions of SET proteins in monocot species. Maize, as one of the

Mol Biol Rep

most important crop species in the world, has become one of the important model monocot species for functional genomic analysis. Although previous study has reported that some SET domain genes have been characterized in maize [30], analysis of the entire SET domain gene family was not completed following maize (B73) genome sequencing project. The availability of the maize genome sequences has provided an excellent opportunity for wholegenome annotation, classification and comparative genomics research [34]. In the present study, we searched for all nonredundant sets of Zmset genes and predicted their presumed structures. The results of this work provide a foundation to better understand functional and evolutionary history of the SET gene family in angiosperms.

Materials and methods In silico identification of Zmset genes Maize genome sequences were downloaded from http://www. maizesequence.org/index.html. A local BLAST database from the maize complete genome nucleotide sequences or protein sequences was subject to be constructed by DNATOOLS software. The Hidden Markov Model (HMM) profile of the SET domain (PF00856) was extracted from Pfam database (http://pfam.sanger.ac.uk/) [35]. This HMM profile was employed as a query to identify all SET-containing sequences in maize by searching SET domain sequence against the B73 maize sequencing database using BlastP program (P value = 0.001). This step was crucial to identify as many similar sequences as possible. Moreover, the starting locations of all candidate SET genes on each chromosome were acquired by TBLASTN (P value = 0.001). Through this method, the physical locations of all candidate SET genes were confirmed and the redundant sequences with the same chromosome location were rejected from the SET candidate list. Subsequently, the Pfam and SMART (http://smart.emblheidelberg.de/) [36] were used to further determine each candidate SET domain-containing protein as a member of the SET gene family. A distinctive name for each of SETs identified in maize was given according to its position from the top to the bottom on the maize chromosomes one to ten. Some basic physical and chemical parameters of each predicted gene was calculated by online Protparam tool (http://www.expasy. org/tools/protparam.html). Sequence alignment and phylogenetic analysis Predicted gene coding sequences were determined using tBlastn and manual comparisons of Clustal-W-aligned genomic sequences, cDNA sequences and predicted coding sequences. All protein sequence alignments were made

using Clustal-W [37]. Phylogenetic analysis was performed with the MEGA v4.0 program [38] by the neighbor-joining method [39] and 1,000 bootstrap replicates were performed. Protein domains were analyzed by scanning protein sequences against the InterPro protein signature database (http://www.ebi.ac.uk/InterProScan) with the InterProScan program. Unless otherwise stated, domains were defined according to Pfam predictions (http://www. sanger.ac.uk/Software/Pfam/). Analysis of conserved domains The conserved domain divergence among SET domain genes in maize was further assessed by a complete amino acid online sequence analysis using SMART (http://smart. embl-heidelberg.de) program and Pfam database. SET domain gene duplication events in maize were also investigated. All of the confirmed SET domain genes from the maize genome were aligned using Clustal-W and calculated using MEGA v4.0 [40]. Gene duplication was defined according to the following criteria [40, 41]: (1) the length of alignable sequence cover [80 % of the longer gene; and (2) the similarity of the aligned regions [70 %. Chromosomal localization of Zmset genes The physical locations of SET domain genes were determined by initially confirming the starting positions of these candidate genes from the maize genome. The positions of maize SET domain genes were subject to online analysis using the TBLASTN program (P value = 0.001) (http://www.maize sequence.org/blast) using predicted coding sequences as query sequences. Through this method, the physical locations of all candidate SET domain genes were confirmed and the redundant sequences with the same chromosome location were rejected from the SET domain candidate list. MapInspect software was subsequently used to draw the location images of maize SET domain genes (http://www.plantbreeding.wur.nl/ UK/software_mapinspect.html). EST expression profile analysis of Zmset genes in silico The analysis of Zmset expression profiles was accomplished by searching the maize dbEST database (http://www.ncbi. nlm.nih.gov/dbEST/) and finding expression information provided at the Web sites. Maize expression data were first obtained through blast searches against the maize dbEST database downloaded from NCBI by conducting the DNATOOLS Blast program. Searching parameters were as follows: maximum identity[95 %, length[200 bp and E value \10-10. In addition to the maize EST database, maize expression data were also extracted from the Maize Assembled Genomic Island (MAGI) (http://magi.

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plantgenomics.iastate.edu/) and the plant genomic database (Plant GBD) (http://www.plantgdb.org/) including EST, cDNA and PUTs (Plant-GDB unique transcripts). Plant materials and stress treatment Maize (Zea mays L. inbred line B73) plants were grown in a greenhouse at 28 °C with a photoperiod of 15 h light and 9 h dark. Three-week-old seedlings were subjected to two abiotic stress treatments. Drought stress was induced by 15 % PEG-6000 (polyethylene glycerol), and seedling leaves were sampled at 24 h after the treatment. Seedling roots were submerged in 0.15 M NaCl solution for salt stress, and seedling leaves were sampled at 8 h after the treatment. The controls were treated with fresh water. Semi-quantitative RT-PCR analysis Total RNA was isolated from the seedlings using Trizol reagent according to manufacturer’s directions (Invitrogen, USA), followed by DNase I treatment to remove any genomic DNA contamination. First-strand cDNA was synthesized using oligo (dT) primer and Superscript II reverse transcriptase (Invitrogen). As a control, reactions were run in parallel that excluded reverse transcriptase. To examine the expression patterns of these predicted genes in maize and to further confirm their stress responsiveness to abiotic stresses such as drought and salt, the class V subfamily, all 18 genes were subjected to semi-quantitative RT-PCR using specific primers designed using Primer 5.0 software. To adjust for RNA quality and differences in cDNA concentration, we amplified actin as an internal control with the following primers: ZmActin-F (50-ATGGCTGACGGTGAG-30) and ZmActin-R (50-TTAGAA GCACTTCCG-30). These genes were amplified from first-strand cDNA using Taq polymerase (Promega) on a thermal cycler (Tpersonal 48; Biometra, Germany), with the following profile: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55.5–64 °C for 45 s, polymerization at 72 °C for 45 s, and final elongation at 72 °C for 5 min. Each PCR pattern was verified by triple replicate experiments; mixture without template was used as negative control and maize actin DNA fragment as positive control for each gene amplified. A 3-lL aliquot of the reaction was separated on 1 % agarose gel.

Results Isolation and characterization of Zmset genes HMM analysis identified 43 SET domain genes encoding ZmSET proteins in the maize genome. These loci were

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confirmed as Zmset genes in the maize genome on the basis of a predicted SET domain from the putative polypeptide sequence. This number is similar to those present in Arabidopsis and rice (47 and 43, respectively) and can also be found in the chromatin database (Table 1). As shown in Table 2, the length of the Zmset open reading frames (ORFs) varied from 372 bp for Zmset38 to 5,448 bp for Zmset26, with the respective coding potential of 123 and 1,815 amino acids. Moreover, the basic physical and chemical parameters of encoded protein, genomic and chromosome location of each gene have also been revealed in Table 2. Similar to Arabidopsis and rice, although all of the SET domain genes encode the conserved SET domain, their sequences elsewhere are highly diverse. Through aligning all the Zmset genes with all known Arabidopsis and rice SET domain genes, we have carried out a classification of maize SET domain genes. Based on their relationships with Arabidopsis and rice SET domain genes, the 43 Zmset genes were divided into seven distinct classes, including class I (3genes), class II (6 genes), class III (6 genes), class IV (6 genes), class V (18 genes), class VI (5 genes) and class VII (3 genes). The number of each type in this classification is also similar to those present in Arabidopsis and rice (Table 2). Phylogenetic and structural analysis of Zmset genes The predicted protein sequences of all the Zmset genes were used to generate an unrooted phylogenetic tree (Fig. 1). The unrooted tree categorized the Zmset genes into seven major groups (Class I–VII) with well-supported bootstrap values. Among 43 Zmset genes, there are fifteen gene pairs, and all of them had strong bootstrap support ([94 %), with the exception of Zmset5/Zmset13, Zmset26/ Zmset9 and Zmset35/Zmset34. Subsequently, an exon– intron structure analysis was performed to support the phylogeny reconstruction. The schematic structures revealed that each coding sequence of Zmset gene is disrupted by one or more exons. The SET genes within the

Table 1 Numbers of SET domain genes in the maize, rice and Arabidopsis genomes Category

Maize

Arabidopsis

Rice

Class I

3

3

2

Class II

6

5

6

Class III

6

7

6

Class IV

2

2

2

Class V

18

15

16

Class VI

5

5

5

Class VII

3

10

6

43

47

43

Total number

Mol Biol Rep Table 2 Basic information of SET domain genes of maize Serial no.

Gene name

Accession number Ensembl transcript

Genome location Coordinates (50 –30 )

ORF length (bp)

Protein

1

Zmset1

GRMZM2G091916_P01

14509271.0.14511175

1,635

544

2

Zmset2

GRMZM2G043484_P02

49406199.0.49416182

2,670

898

3 4

Zmset3 Zmset4

GRMZM2G318803_P01 GRMZM2G451374_P01

201626846.0.201631389 211265879.0.211271584

2,163 801

720 266

5

Zmset5

GRMZM2G074094_P02

220005421.0.220009243

3,486

1,161

6

Zmset6

GRMZM2G080462_P02

242140834.0.242149635

522

173

19240.89

4.43

1

VI

7

Zmset7

GRMZM2G164277_P02

7523654.0.7528053

1,410

469

51995.69

4.7

2

VI

8

Zmset8

GRMZM2G021044_P01

21283319.0.21286542

2,661

886

97155.5

6.81

2

V

9

Zmset9

GRMZM2G147619_P03

51820268.0.51827266

1,542

513

57252.8

4.96

2

II

10

Zmset10

GRMZM2G401062_P01

53789049.0.53794150

1,920

639

70741.13

6.01

2

VII

11

Zmset11

GRMZM2G025924_P01

169539961.0.169542455

2,136

711

77815.14

5.37

2

V

12

Zmset12

GRMZM2G033694_P03

173776072.0.173793316

1,032

343

39786.41

7.47

2

II

13

Zmset13

GRMZM2G300955_P01

176724623.0.176730005

3,927

1,308

140185.6

6.09

2

V

14

Zmset14

GRMZM2G409224_P03

198096984.0.198115319

4,776

1,591

176556.4

8.77

2

III

15

Zmset15

GRMZM2G105869_P02

199386714.0.199389560

1,020

339

7.42

2

V

16

Zmset16

GRMZM2G085266_P01

3200782.0.3209083

2,916

971

7.3

3

III

17

Zmset17

GRMZM2G416913_P01

103815531.0.103817630

2,010

699

77154.03

7.38

3

V

18 19

Zmset18 Zmset19

GRMZM2G336909_P01 GRMZM2G139710_P01

156800688.0.156815457 187028837.0.187033469

2,007 2,301

668 766

74005.23 83842.79

8.2 5.58

3 3

V V

20

Zmset20

GRMZM2G042442_P01

48342386.0.48347154

2,220

739

81757.99

5.67

4

V

21

Zmset21

GRMZM2G067019_P01

136070311.0.136101261

1,257

418

45782.36

8.03

4

II

22

Zmset22

GRMZM2G360389_P01

141522853.0.141526357

702

233

23

Zmset23

GRMZM2G172427_P01

160924313.0.160939504

4,806

1,601

24

Zmset24

GRMZM2G457881_P01

11946345.0.11954653

1,449

482

25

Zmset25

GRMZM2G305124_P01

71843738.0.71845004

1,164

387

26

Zmset26

GRMZM2G352431_P02

172565584.0.172579182

5,448

1,815

27

Zmset27

GRMZM2G125432_P01

201593842.0.201608408

4,386

28

Zmset28

GRMZM2G157820_P02

79459102.0.79469316

29

Zmset29

GRMZM2G117458_P01

155462838.0.155481947

30

Zmset30

AC233961.1_FGP001

31

Zmset31

32

Zmset32

33 34 35

Length (a.a.)

Chr. Mol. Wt. (Da) 58929.18 100908.5 79349.26 30197.74 126175.4

35641.68 108592.3

26590.52

Type

PI 7.15

1

VI

8.49

1

I

5.81 8.72

1 1

V III

9.82

1

V

8.64

4

V

6.34

4

V

54587.06

7.96

5

VI

42687.97

9.35

5

IV

198447.4

6.5

5

II

1,461

163955.3

6.36

5

V

2,796

931

103768.5

8.85

6

I

2,025

674

74125.69

7.92

6

V

20244021.0.20246591

2,571

856

93443.85

8.56

7

V

GRMZM2G034288_P01

38205507.0.38218647

2,049

682

GRMZM2G013794_P02

72242504.0.72287055

2,949

982

Zmset33

GRMZM2G130910_P01

84844084.0.84855290

1,014

337

Zmset34 Zmset35

GRMZM2G418752_P01 GRMZM2G092131_P01

110914563.0.110919344 144602194.0.144604805

1,470 1,452

489 483

36

Zmset36

GRMZM5G807767_P03

76493336.0.76498313

1,485

37

Zmset37

GRMZM2G140577_P01

119829269.0.119832857

2,088

38

Zmset38

GRMZM2G179814_P01

147725068.0.147726498

372

123

14267.33

6.17

8

III

39

Zmset39

GRMZM2G149587_P02

156906971.0.156912759

1,146

381

43002.49

8.73

8

IV

40

Zmset40

GRMZM2G054380_P04

160240039.0.160254725

2,310

769

84998.07

8.55

8

V

41

Zmset41

GRMZM5G875502_P02

135256308.0.135271042

2,685

894

99979.37

8.47

9

I

42

Zmset42

GRMZM2G473138_P01

4015808.0.4020018

2,133

710

79991.96

8.9

10

III

43

Zmset43

GRMZM2G052328_P01

63399792.0.63403707

1,215

404

43276.4

4.58

10

VI

179432.6

74711.95

5.73

7

V

6.96

7

III

38941.46

7.77

7

II

54239.5 54094.6

4.92 5.3

7 7

VII VII

494

56808.33

5.18

8

III

695

76950.03

7.92

8

V

111093.4

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same groups of the phylogenetic tree all showed similar exon–intron structures (Fig. 1). To further determine the phylogenetic relationships between SET domain genes from different species and to assess the evolutionary history of these families among maize, rice and Arabidopsis, we performed an analysis by using the highly-conserved SET-domain region of each SET protein sequence from these plants and constructed an unrooted neighbor-joining phylogenetic tree (Fig. 2). The unrooted tree categorized the Zmset genes into seven major groups (Class I–VII) with well-supported bootstrap values. This result is consistent with the latest report [2]. According to the previous reports, the class I was further subdivided into three subclasses: the Arabidopsis proteins CLF (SDG1), MEA (SDG5) and EZA/SWN (SDG10). Interestingly, in maize, we only found one homolog of CLF (Zmset28) and two of EZA/SWN (Zmset2 and Zmset41, respectively), and no homologs of MEA were found in this study. The same result was also shown in another monocot rice. Furthermore, the unrooted neighbor-joining phylogenetic tree revealed that similar numbers of members are grouped in other six classes including the largest class (Class V) among these three distinct plants. Analysis of conserved domains in maize SET proteins In order to further analyze the architecture of maize SET domain proteins and reveal the structural differentiation of proteins classified in a particular class, all conserved domains including the SET domain were characterized by

Fig. 1 Phylogenetic relationship and gene structure of the 43 predicted maize SET domain proteins. a The unrooted tree was constructed with MEGA4.0 program using the full-length amino acid sequences of the 43 maize SET domain proteins. The bootstrap values are indicated at the branches in black numbers, and the proteins were named according to their gene codes. b Exons and introns are indicated by white boxes and single lines, respectively. Thick gray lines represent the untranslated regions (UTRs). The length of each SET domain gene can be estimated using the scale at the bottom

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using Pfam database and SMART program as shown in Fig. 3. In class I, all of three maize SET proteins contain an N-extreme SANT domain (SWI3, ADA2, N-CoR, and TFIIIB DNA-binding domains) in addition to the conserved SET domain. All class II maize SET domain proteins have a conserved AWS domain located just N-terminal of the SET domain, which is a subdomain of the PreSET domain that contains several highly conserved Cys residues [30]. Furthermore, another conserved PostSET domain following C-terminal of the SET domain exists in these proteins (except ZmSET4). The Class III proteins belong to a highly diverse subfamily with the existence of four orthology groups. In maize, ZmSET32, homology of the Arabidopsis group III-1 proteins (SDG27 and SDG30), contain a similar arrangement of domains including a PWWP domain, an FYR domain that is composed of an FYR-C terminal portion and an FYR-N terminal portion that often occur near each other but can be separated [42], and two PHD domains (Fig. 3). A second orthology group (III-2) of the class III plant SET domain proteins includes the Arabidopsis proteins SDG14, SDG16 and SDG29 usually contain a PWWP domain, two PHD domains, and a PostSET domain in addition to the SET domain [30]. However, in this study, we found that ZmSET16 has three PHD domains and ZmSET38 loses all N-terminal PWWP and PHD domains of the SET domain. Furthermore, another two orthology groups (III-3 and III-4) only own a homology protein (ZmSET42) in maize. The class IV SET domain proteins, ZmSET25 and ZmSET39, only include an N-terminal PHD domain and a conserved SET domain. The class V proteins are the largest group of

Mol Biol Rep Fig. 2 Phylogenetic relationships of maize, rice and Arabidopsis SET domain proteins. The tree was constructed by the NeighborJoining method with MEGA program 4.0 using the conserved SET-domain region of maize, rice and Arabidopsis SET domain proteins. The accession numbers of the maize and Arabidopsis SET domain sequences are shown in Table 2 and Springer et al. [30], respectively

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Mol Biol Rep b Fig. 3 Schematic structures of maize SET domain proteins. Sche-

matic structures of 43 SET domain proteins identified in maize are shown with names of all the members on the left side of the figure. Different domains are indicated by different boxes denoted at the right bottom corner. Domain abbreviations: SET, (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain; SANT, SWI3, ADA2, N-CoR and ‘‘TFIIIB’’ DNA-binding domains; AWS, associated with SET domains; PostSET, Cysteine-rich motif following a subset of SET domains; PHD, PHD zinc finger; PWWP, domain with conserved PWWP motif; FYRN, ‘‘FY-rich’’ domain, N-terminal region; FYRC, ‘‘FY-rich’’ domain, C-terminal region; SRA, SET and RING finger associated domain; PreSET, N-terminal to some SET domains; ZnF_C2H2, zinc finger. The proteins were grouped manually according to the number of SET domain and the distribution of other conserved domains

and C-terminal of the SET domain, respectively. Springer et al. [30] divided the Arabidopsis class V SET domain proteins into seven orthology groups V-1, V-2, V-3, V-4, V-5, V-6 and V-7. Among them, the orthology groups V-1, V-2, V-3 and V-5 are all YDG(SRA)/PreSET/SET/PostSET domain proteins, whereas the orthology groups V-4, V-6, and V-7 all lack the YDG(SRA) domain. In this study, except the orthology group V-5, all of 18 ZmSET proteins of class V fall into six orthology groups (Fig. 3). Of them, the orthology group V-7 proteins, ZmSET23 and ZmSET27 have 2–4 copies of another ZnF-_C2H2 domain in N-terminal of PreSET and SET domains, respectively. Finally, another two classes VI and VII lack the additional domains characterized in other classes of plant SET proteins. Chromosomal localization and gene duplication

SET domain proteins in plants to date, which is also the only class of SET domain proteins characterized by the presence of both PreSET and PostSET domains located in N-terminal

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The physical locations of SET domain genes in maize were investigated by analysis of genomic distribution on chromosomes. The 43 SET domain genes were found to be distributed unevenly across all the ten chromosomes in the maize genome (Fig. 4). The number of Zmset genes appearing on each chromosome had a wide range. The chromosome two contains the maximum number of genes (nine), followed by the chromosomes one and seven (six, respectively). By contrast, the chromosomes six, nine and ten all have only one or two Zmset genes, respectively. To elucidate the expanded mechanism of the Zmset gene family, gene duplication events including tandem and segmental duplications, which are thought to have occurred during the process of evolution, were investigated in maize. Based on the phylogenetic analysis and the chromosomal distribution of the Zmset genes, seven gene pairs (Zmset29/ Zmset37, Zmset11/Zmset31, Zmset18/Zmset40, Zmset3/ Zmset20, Zmset23/Zmset27, Zmset12/Zmset33 and Zmset2/ Zmset41) were identified to be involved in the segmental duplication events (Fig. 4). Among the seven segmental duplication events, there were five gene pairs that belong to high frequency of segmental duplication occurred among

Mol Biol Rep Fig. 4 Chromosomal locations of maize SET domain genes on all 10 chromosomes. Chromosome numbers are shown at the top of each vertical gray bar. The gene names on the left side of each chromosome correspond to the approximate locations of each SET domain gene. The segmental duplication genes are connected by dashed lines. The scale on the left is in megabases

the class V SET domain genes. Another two gene pairs (Zmset12/Zmset33 and Zmset2/Zmset41) exist in the class II and I, respectively. Furthermore, no tandem duplicated gene pair was found in this study. EST expression profile of Zmset gene family in silico The NCBI EST database provides a large number of ESTs generated from the maize FLcDNA project. These EST data mainly consist of mixed or individual tissue and organ types released by the maize FLcDNA project [43]. In this study, maize Zmsets expression patterns were studied using corresponding EST database with known Zmsets coding sequence, resulted in the assignment of Zmsets to twelve groups on the basis of tissue and organ types (Table 3). In addition, other expression evidence was verified in MAGI and PlantGDB databases (Table 3). After integrating and analyzing all expression data, we found all Zmsets were supported by expression evidence with the exception of the Zmset6 gene. Interestingly, most of these genes were shown to obtain expression evidence from mixed or individual tissue and organ types except husks and root tips. Among them, there were 13 Zmset genes that were revealed

to express in only mixed tissues and to have no tissuespecific expression pattern. The others (21 Zmset genes) were shown to specifically express in one or more tissue and organ types. Furthermore, Zmset duplicated gene pair expression patterns were investigated, only two pairs (Zmset11 and Zmset31, Zmset12 and Zmset33) of seven shared the same expression patterns between the two members of each gene pair. In the other five duplicated gene pairs, two paralogs of each gene pair exhibited dissimilar expression patterns. For example, Zmset3 was detected in silks and tassel, however, its paralogue gene Zmset20 appeared to have tissue-specific expression pattern in shoot tips and leaf. The expression pattern assay of all members of the class V subfamily of maize SET domain genes under stress treatment To confirm these predicted genes and understand their expression profiles under various stress conditions, two abiotic stress treatments (PEG for drought stress and NaCl for salt stress) were investigated. Semi-quantitative RTPCR analyses on RNA isolated from maize leaves was

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Fig. 5 Semi-quantitative RT-PCR analysis of maize SET genes (the class V subfamily) under stress treatments. Total RNA was extracted from the 21-day-old seedlings germinated on wet filter paper soaked with distilled water and grown in a growth chamber. Either fresh water (1), 15 % PEG (2) or 0.15 M NaCl (3) was applied 24 h before harvest. An amplified maize actin gene was used as an internal control

performed (Fig. 5). The results revealed that these genes were differentially expressed in the leaves under either normal condition (control) or stress conditions (15 % PEG or 0.15 M NaCl treatment). On the basis of the brightness of the bands, most of the expressed genes showed increased or decreased expression levels in maize leaves by PEG or NaCl treatment, whereas six genes including Zmset5, Zmset13, Zmset15, Zmset17, Zmset20 and Zmset23 showed no obvious difference in the leaves of maize under these two stress conditions. Moreover, five members including Zmset3, Zmset27, Zmset29, Zmset31and Zmset40 exhibited differential expression with decreased expression levels in maize leaves by PEG treatment and increased expression levels by NaCl treatment. Furthermore, there were five genes that exhibited differential expression only under PEG treatment and no difference under NaCl treatment. These results demonstrated that these predicted genes exhibit different expression levels in stress treatments.

Discussion The histone methylation at specific lysine residues has been demonstrated to control multiple developmental processes as one of important epigenetic mechanisms in plants. In this study, a comprehensive set of 43 nonredundant SET domain proteins were identified and characterized from the

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latest version of the maize (B73) genome, which has been completely sequenced and can provide more precise and complete genome databases in maize. Interestingly, although the maize genome is approximately sixfold larger than rice, we found that both maize and rice have the same number of SET domain genes. This further revealed that their gene number is similar and their genetic map organization is highly conserved in these two species during the evolutionary process. Furthermore, phylogenetic analysis provided insights into the evolution of gene family members and gene multiplicity in maize. In our investigation of conserved SET domain proteins, we found that maize SET domain genes were classified into seven distinct classes, which is also similar to those present in Arabidopsis and rice. This further demonstrated that maize shares a close relationship with Arabidopsis and rice during the evolutionary process. Furthermore, phylogenetic analysis of SET domain genes in maize, rice and Arabidopsis indicated that Zmsets are more closely allied with Ossets than Atsets, consistent with the evolutionary relationships among maize, rice and Arabidopsis. The fact that all seven classes (I–VII) identified in maize, rice and Arabidopsis genes implies that the SET domain genes originated prior to the divergence of monocots and dicots. It is suggested that the expansion of these SET domain genes following divergence of monocots and dicots. According to the previous reports, in Arabidopsis, the class I (Enhancer of zeste [E(Z)] homologs) is related with H3K27, the class II (the ASH1 homologs) with H3K36, classes III (the trithorax homologs and related proteins) and IV (Proteins with a SET and a PHD domain) with H3K4, the class V (Suppressor of variegation [Su(var)] homologs and relatives) with H3K9 and the class VII (RBCMT and other SET-related proteins) with the methylation of nonhistone targets [1, 2]. However, the function of class VI (Proteins with an interrupted SET domain) has yet remained unclear in plants. These results further revealed that diverse mechanisms could be involved in shaping the function and regulation of these SET domain proteins [2]. In this study, we found no homologs of MEA in the class I of SET domain genes in monocot plants including maize and rice. A possible excuse is the functional diversification of homologs of MEA in the class I of SET domain genes occurred after the divergence of monocots and dicots *200 million years ago. Secondly, although the SET domain proteins encoded in the maize genome are grouped into all of the classes identified in other species, the ZmSET proteins of the largest class V only fall into six orthology groups, different from the Arabidopsis class V SET domain proteins into seven orthology groups (V-1–7). Thus, the maize genome lacks the homologs of the orthology group V-5 in Arabidopsis class V SET domain proteins.

?

?

?

endosperm

?

Seed

Mixed

?

?

?

?

?

?

?

?

?

?

? ?

?

?

Zmset34

Zmset33

Zmset32

Zmset31

?

?

?

?

?

?

?

?

?

?

?

Zmset29

Zmset30

1

?

Zmset28

2

2

4

4

7

22

1

5

Zmset27

0 ?

2

Zmset26

?

4 4

8

9

1

5

Zmset25

Zmset24

?

?

?

Zmset22 Zmset23

? ?

?

Zmset20

?

?

Zmset19

Zmset21

?

Zmset18

16

?

Zmset17

?

3

Zmset16 ?

0 ?

Zmset15

?

?

2

2 0

?

?

Zmset14

Zmset13

Zmset12

4

?

Zmset11

1 0

?

Zmset10

Zmset9

10 1

Zmset7 Zmset8 ?

0

Zmset6 ?

4

5

3

6

?

?

embryo

0

?

?

Seeding

Zmset5

?

Root tips

Number of ESTs in dbEST

Zmset4

?

Leaf

?

?

Shoot tips

Zmset3

Tassel

?

Ear ?

Husks

Zmset2

Silks

Tissue and organ type (NCBI)

Zmset1

Gene

Table 3 Expression analysis of Zmset genes in silico

?

?

?

?

?

?

?

?

?

?

?

? ?

?

?

?

?

?

?

?

?

?

?

?

?

?

? ?

?

?

?

?

?

MAGI

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

EST

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

? ?

?

?

?

cDNA

PlantGDB

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

? ?

?

?

?

?

PUTs

Mol Biol Rep

123

?

? ? ? ? 3 ? Zmset43

? 0 Zmset42

?

? ?

? ? ? 8

? ?

? ? ?

?

123

? Zmset41

Zmset35

? expressed, blank not expressed

Tassel Ear Husks Silks

Tissue and organ type (NCBI) Gene

Table 3 continued

Shoot tips

?

Leaf

Root tips

Seeding

?

embryo

?

endosperm

?

?

?

?

? Zmset40

6

? Zmset39

1

?

?

? ? ? ?

? 5

17

?

? Zmset37

Zmset38

?

? ?

? ?

? ?

? 0

9 ?

Seed

Mixed

Number of ESTs in dbEST

Zmset36

cDNA EST

MAGI

PlantGDB

PUTs

Mol Biol Rep

Furthermore, the characterization of conserved domains in maize SET proteins also contributes to elucidate the mechanism diversity in shaping the function and regulation of these SET domain proteins. The conserved SET domain has been demonstrated to play a key role in fulfilling the function of histone lysine methylation at specific lysine residues targeted by these SET domain proteins. The other domains present in many of the plant SET domain proteins have also been revealed to exert some important functions in protein–protein interactions. This further suggests that the plant putative histone methyltransferases may act in protein complexes. For instance, the presence of PHD and PWWP domains has been demonstrated to play an important in constructing protein complexes [13]. The previous reports have indicated that the PHD domain is composed of a putative zinc finger that is involved in mediating protein– protein interactions [44], and that the PWWP domain is also involved in mediating protein–protein interactions [45]. In addition, gene expression in silico data from EST databases plays an increasingly vital role in providing gene expression research information. This facilitates the identification of gene function and future functional genomics studies in plant growth and development [43]. In this study, several approaches were employed for maize SET domain gene expression patterns in different tissues or organs. EST expression data mining revealed that these newly identified genes exhibited distinct expression patterns in different tissues or organs. One explanation is that some of these investigated genes may have temporal and spatial expression pattern, which varies with tissue types, developmental stages or genotypes of maize. Furthermore, the expression data revealed that the majority of duplicated Zmset gene pairs exhibited diverse expression patterns between two members. It suggested that functional diversification of the surviving duplicated genes is a major feature of the long-term evolution [46]. Furthermore, the expression analyses of semi-quantitative RT-PCR in this study showed that maize Zmset genes (the Class V subfamily) exhibited different expression levels under two different abiotic stress treatments. For 12 candidate genes, the expression levels increased or decreased after applying 15 % PEG or 0.15 M NaCl treatment than for controls, suggesting that these genes might play important roles in plant histone methylation regulation, especially those showing strong response to the two abiotic stress conditions in this study. In contrast, another six genes (Zmset5, Zmset13, Zmset15, Zmset17, Zmset20 and Zmset23) that showed no obvious difference in the leaves of maize under these two stress conditions, had a high possibility to contribute to maize histone methylation regulation by only expressing under specific conditions or in specific tissues other than seedling leaves, and these remain to be further confirmed experimentally.

Mol Biol Rep

Conclusions At present, a comprehensive overview of the SET domain gene family repertoire within the maize draft genome has been presented in this study. Based on structural characteristics and a comparison of the phylogenetic relationships among maize, rice and Arabidopsis, all 43 ZmSET proteins can be phylogenetically grouped into seven major classes (Class I–VII), and these distinct classes may be involved in histone methylation at specific lysine residues, which determines the mechanism diversity in shaping the function and regulation of these SET domain proteins and exerts multiple diverse roles in multiple developmental processes in plants. Further phylogenetic analysis revealed divergent expansion patterns of SET domain gene families in distinct classes. Our results suggest that whole genome and chromosomal segment duplications largely contributed to SET domain gene family expansion in maize. Furthermore, our computational expression analyses suggest that many maize SET domain genes play functional developmental roles in multiple tissues. The transcripts of the eighteen genes (the Class V subfamily) were detected in the leaves by two different abiotic stress treatments using semiquantitative RT-PCR, which demonstrated that these genes exhibited different expression levels in stress treatments. Overall, our study will serve to better understand the complexity of the maize SET domain gene family and also be beneficial for future experimental research to further unravel the mechanisms of epigenetic regulation in plants. Acknowledgments This study was supported by grants from the China Postdoctoral Science Foundation (No. 2012M521212) and the Anhui Provincial Natural Science Foundation (No. 1308085MC44) and the Anhui Provincial University Natural Science Research Key Project (No. KJ2013A132) and the Anhui Postdoctoral Science Foundation. We wish to thank the anonymous reviewers for their helpful comments on this manuscript. We also acknowledge Professor Yilong Xi and Professor Beijiu Cheng for critical reading of the manuscript.

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