Identification and characterization of Lateral Organ Boundaries Domain genes in mulberry, Morus notabilis.

Genes from the plant specific Lateral Organ Boundaries Domain (LBD) family encode transcriptional regulators that have a variety of functions in various physiological and developmental processes. In the present study, 31 LBD genes were identified in the mulberry genome. The genome features of all MnLBD genes and phylogenetic studies with Arabidopsis LBD protein sequences, accompanied by the expression analysis of each of the Morus LBD genes provide insights into the functional prediction of mulberry LBDs. The genome-wide surveys of the current mulberry genome have resulted in the identification of catalogs of MnLBD genes that may function in the development of leaf, root, and secondary metabolism in Morus sp.

Introduction

The first Lateral Organ Boundary (LOB) gene was identified in Arabidopsis thaliana based on the expression pattern of an enhancer trap insertion and was found to be expressed at the boundaries of lateral organs during vegetative and reproductive plant development (Shuai et al., 2002). An important outcome of the above mentioned work was the discovery of a new conserved protein domain, the LOB domain, a plant specific domain present in 42 other Arabidopsis proteins (Shuai et al., 2002). In A. thaliana, the conserved LOB domain (LBD) contains approximately 100 amino acids. Furthermore, all 42 LBDs were further divided into two sub classes (Class I and II) according to their sequences. All LBD proteins were previously thought to contain a conserved C block with C-x(2)-C-x(6)C-x(3)-C motif in the N terminus, and it was identified as a zinc finger-like domain, which may play an important role in DNA binding (Shuai et al., 2002, Matsumura et al., 2009). However, Class I LBDs were also found to have an additional GAS motif and a L motif L-x(6)-L-x(3)-L-x(6)-L. The L motif is a leucine zipper-like coiled-coil motif, which is possibly involved in protein dimerization (Shuai et al., 2002). Experimental evidence showed that LBD genes encode a class of DNA-binding transcription factors and the LOB domain can interact with the basic helix–loop–helix family of transcription factors (Husbands et al., 2007). Recent studies have suggested that LBD genes have a broad range of functions. An LBD-like gene in rice, ARL1, expressed in lateral and adventitious root primordia, tiller primordia, vascular tissues, scutella, and young pedicels, is an auxin-responsive factor involved in adventitious root formation (Liu et al., 2005). Genes LBD16, 29, 18 in A. thaliana mediate lateral root formation (Okushima et al., 2007, Lee et al., 2009, Lee et al., 2013). LBD33 is involved in lateral root organogenesis by forming a dimer with LBD18 and the LBD18/LBD33 dimer transcriptionally activated Arabidopsis E2Fa to initiate cell cycle in the lateral root (Berckmans et al., 2011). Serrano-Cartagena (1999) found that the gene, asymmetric leaves 2 (AS2, termed LBD6 later) was relative to leaf development. The abnormal expression of LBD6 and LBD36 genes causes hyponastic leaves, downward-pointing flowers, and decreased apical dominance as shown by the gain-of-function mutant by activation tagging and loss-of-function mutant in Arabidopsis (Nakazawa et al., 2003, Chalfun-Junior et al., 2005). Members of the LBD proteins are also involved in plant secondary metabolism, such as class II LBDs 37, 38 and 39, which act as negative regulators of anthocyanin biosynthesis in A. thaliana (Rubin et al., 2009). The expression patterns of several of the 42 Arabidopsis LBD genes change in association with phytohormones. The most prominent among these is AtLBD40, whose expression was down regulated by gibberellin (Zentella et al., 2007).

Mulberry (Moraceae: Rosales) is a perennial woody plant and is widely planted for silkworm rearing (Ganga, 2003). It tolerates of pruning and bud growing well in the following spring (Yamashita and Fujino, 1986). Moreover, mulberry has a strong root system (Olson et al., 2003), which enables it to withstand natural calamities such as desertification, drought, and flood. Numerous active secondary metabolites have been detected in the sorosis, root bark, and leaves of mulberry, which have medicinal value especially in traditional Chinese medicine, and are used for the treatment of diabetes, arthritis, and rheumatism (Nomura, 1988, Sun et al., 2001, Singab et al., 2005).

The genome of mulberry was recently sequenced and is available in the Morus database (http://morus.swu.edu.cn/morusdb/). This data provides an opportunity to analyze the mulberry LBD genes. LBD proteins are plant specific transcription factors and play important roles in almost every aspect of plant development (Majer and Hochholdinger, 2011). Therefore, the identification of mulberry LBD genes, revealing their genomic structure, and analyzing their transcriptional profiles will contribute greatly to understanding their role in mulberry development.

Materials and methods

Identification of the mulberry LBD family genes

The Morus database was used (http://morus.swu.edu.cn/morusdb/). Forty-two Arabidopsis LBDs were downloaded from the Plant Transcription Factor Database (http://planttfdb.cbi.edu.cn/) (Husbands et al., 2007). The Hidden Markov model (HMM) profile for the LBD family (DUF260, Pfam number: PF0319) was obtained from the Pfam (http://pfam.sanger.ac.uk/) (Punta et al., 2012). Two methods were used to search against the mulberry peptide database. First, all 42 Arabidopsis LBDs were used as queries to search by BLASTP (Altschul et al., 1997) at an e-value of 1e-10. The redundancies were excluded. Secondly, the HMM profile of the LOB domain (Accession no. DUF260) was downloaded from the Pfam database (http://www.sanger.ac.uk). This domain was used as a query to blast against the mulberry peptide database with the BLASTP program. The predicted genes obtained in two methods were examined and corrected by the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/) (Letunic et al., 2012) and GENSCAN Web Server (http://genes.mit.edu/GENSCAN.html) (Burge and Karlin, 1997). Information regarding CDS length, amino acids number, molecular weight, and isoelectric point of protein were downloaded from TIGR release 4. The gene annotations in Table 1 were searched using protein blast on NCBI (http://ncbi.nlm.nih.gov) and they all based on the Arabidopsis LBD members. The predicted functions for some of the genes have been described in Arabidopsis in previous studies.

Table 1

List of 31 LBD genes identified in mulberry and their sequence characteristics (bp, base pair; aa, amino acids) coupled with the annotation results.

Name	Accession no.	Location	Gene			Protein			Predictions
Strand	Introns no.	CDS (bp)	aa	Mw(D)	pI	Annotation	Functions	References
MnLBD1	Morus014650	Scaffold6: 187,049–187,238:187,612–188,168	+	1	747	248	26,920.5	8.31	LOB domain-containing protein 41-like	Leaf dorsoventral determination	(Meng, 2009)
MnLBD2	Morus020555	Scaffold39: 444,787–445,296	−	0	510	169	18,689	6.98	LOB domain-containing protein 25-like	Auxin signaling and photomorphogenesis	(Mangeonet 2011)
MnLBD3	Morus009777	Scaffold40: 77,112–77,405:78,023–78,232	+	1	504	167	18,172.6	8.46	LOB domain-containing protein 16-like	Effecting dedifferentiation of pericycle cells, lateral root formation	(Feng, 2012); (Lee et al., 2009); (T Goh,2012)
MnLBD4	Morus009778	Scaffold40: 86,251–86,655:86,797–87,075	−	1	684	227	24,930.8	6.11	LOB domain-containing protein 29-like	Effecting dedifferentiation of pericycle cells, lateral root formation	(Feng, 2012)
MnLBD5	Morus009779	Scaffold40: 96,469–96,909:97,273–97,551	−	1	720	239	26,676.7	6.15	LOB domain-containing protein 29-like	Effecting dedifferentiation of pericycle cells, lateral root formation	(Feng, 2012)
MnLBD6	Morus014552	Scaffold125: 169,868–170,416	+	0	549	182	20,150.5	6.69	Lateral Organ Boundaries-like protein	Lateral organ development	(Shuai et al., 2002)
MnLBD7	Morus014124	Scaffold127: 242,300–242,671:243,592–243,912	+	1	693	230	24,924.5	6.7	LOB domain-containing protein 1-like	Secondary woody growth	(Yordanov, 2010)
MnLBD8	Morus025355	Scaffold139: 290,612–290,890:290,972–291,403	+	1	711	236	26,115.9	4.95	LOB domain-containing protein 33-like	Lateral root organogenesis	(Berckmans et al., 2011)
MnLBD9	Morus025918	Scaffold276: 181,491–181,850:183,993–184,184	−	1	552	183	19,561	8.56	LOB domain-containing protein 4-like
MnLBD10	Morus027282	Scaffold329: 591,410–591,509:593,976–594,130:594,532–595,110	−	2	834	277	30,826.6	7.67	LOB domain-containing protein 22-like
MnLBD11	Morus023484	Scaffold404: 625,335–625,520:638,942–639,277	+	1	522	173	19,816.8	6.06	LOB domain-containing protein 11-like
MnLBD12	Morus013514	Scaffold410: 269,681–269,833:270,199–270,579	+	1	534	177	20,389.9	6.96	LOB domain-containing protein 24-like
MnLBD13	Morus022437	Scaffold594: 218,060–218,153:219,373–219,680:222,234–222,554	−	2	723	240	26,296	9.04	LOB domain-containing protein 31-like
MnLBD14	Morus022439	Scaffold594: 241,631–241,963:244,129–244,527	+	1	732	243	25,276.5	7.1	LOB domain-containing protein 18-like	Tracheary element differentiation; lateral root formation	(Soyano, 2008; (Lee et al., 2009)
MnLBD15	Morus014422	Scaffold710: 370,069–370,467:371,293–371,568	−	1	675	224	24,539.6	5.68	LOB domain-containing protein 1-like	Secondary woody growth	(Yordanov, 2010)
MnLBD16	Morus013233	Scaffold720: 117,943–118,902	−	0	960	319	35,307.6	6.62	LOB domain-containing protein 36-like	Flower development	(Chalfun-Junior et al., 2005)
MnLBD17	Morus015733	Scaffold759: 53,703–53,855:54,737–55,123	+	1	540	179	20,692.5	8.19	LOB domain containing protein 24-like
MnLBD18	Morus006407	Scaffold778: 83,597–83,720:83,820–84,616	−	1	921	306	34,393.2	5.44	LOB domain-containing protein 27-like	Microspore development and asymmetric division	(Oh, 2010)
MnLBD19	Morus005914	Scaffold833: 80,444–80,627:80,720–81,249	+	1	714	237	25,617.1	8.79	LOB domain-containing protein 38-like	Anthocyanin biosynthesis	(Rubin et al., 2009)
MnLBD20	Morus005107	Scaffold847: 75,061–75,190:75,288–75,943	−	1	786	261	28,747.2	5.31	LOB domain-containing protein 22-like
MnLBD21	Morus008640	scaffold1006: 20,558–21,288:21,390–21,579	−	1	921	306	32,916.4	8.42	LOB domain-containing protein 41-like	Leaf dorsoventral determination	(Meng, 2009)
MnLBD22	Morus005283	Scaffold1065: 73,817–74,575	+	0	759	252	27,518.1	7.75	LOB domain-containing protein 36-like	Flower development	(Chalfun-Junior et al., 2005)
MnLBD23	Morus016263	Scaffold1154: 391,145–391,480:394,954–395,133	−	1	516	171	18,920.4	7.67	LOB domain-containing protein 4-like
MnLBD24	Morus009899	Scaffold1198: 190,981–191,532	+	0	552	183	20,024.7	8.69	LOB domain-containing protein 21-like
MnLBD25	Morus003679	Scaffold1252: 33,898–34,340:34,424–34,607	−	1	627	208	22,356.2	6.89	LOB domain-containing protein 38-like	Anthocyanin biosynthesis	(Rubin et al., 2009)
MnLBD26	Morus011165	Scaffold1300: 320,516–320,893:321,188–321,343	−	1	534	177	19,532	6.39	LOB domain-containing protein 12-like	Leaf development	(Nakazawa et al., 2003)
MnLBD27	Morus003750	Scaffold1448: 108,282–108,956	+	0	675	224	24,230	8.25	LOB domain-containing protein 6-like	Maintains shoot meristem and defines lateral organ boundary; leaf and flower development	(Uchida, 2007); (Xu L, 2003); (Xu B, 2008)
MnLBD28	Morus005793	Scaffold1629: 128,982–129,218:129,876–130,028	−	1	390	129	14,329.5	8.8	LOB domain-containing protein 24-like
MnLBD29	Morus002082	Scaffold1681: 56,758–57,467:57,585–57,726	+	1	852	283	31,686.7	5.75	LOB domain-containing protein 27-like	Microspore development and asymmetric division	(Oh, 2010)
MnLBD30	Morus006937	Scaffold2113: 114,567–114,722:115,086–115,469	+	1	540	179	19,931.7	6.28	LOB domain-containing protein 12-like	Leaf development	(Nakazawa et al., 2003)
MnLBD31	Morus000182	Scaffold11171: 854–1037:1146–1588	+	1	627	208	22,764	9.06	LOB domain-containing protein 38-like	Anthocyanin biosynthesis	(Rubin et al., 2009)

Phylogenetic and gene structure analysis

Multiple alignments of LOB-domain protein sequences were performed using the ClustalW program (Chenna, 2003). Phylogenetic trees were constructed using the MEGA 5.0 software (Tamura et al., 2011) and the neighbor-joining (NJ) method with the p-distance and complete deletion option parameters. The reliability of the trees was tested using a bootstrapping method with 1000 replicates. A diagram of exon–intron structures was generated using the online Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) (Guo et al., 2007) with the sites of intron and exon by loading DNA and RNA sequences of mulberry LBD gene family (RNA sequences was shown in Supplementary material 1). The conserved sequence logo was obtained using the online Weblogo platform (http://weblogo.berkeley.edu) (Schneider and Stephens, 1990). The conserved motifs were searched on “Multiple Em For Motif Elicitation” (MEME version 2.2, http://meme.nbcr.net/) using the following parameters, − nostatus − time 7200 − maxsize 60,000 − mod zoops − nmotifs 50 − minw 6 − maxw 50 (Bailey and Elkan, 1994).

Expression analysis of the mulberry LBD family genes

The reads per kilobase of exon model per million mapped reads (RPKM) were used for comparing the differences of gene expression among samples (Bullard et al., 2010). The root, bark, bud, flower, and leaf RPKM value of mulberry LBD genes were retrieved from RNA sequencing data (http://morus.swu.edu.cn/morusdb/). A heat map was created by the Multi Experiment Viewer (Mev, version 4) (Saeed et al., 2003). Data were adjusted using normalize genes/row. Hierarchical clustering was performed using a default parameter.

Results

Identification of LBD genes in the mulberry genome

BLAST program and HMM analysis resulted in 31 mulberry LBD genes. All the 31 MnLBDs contained the LOB domain and the length ranged from 129 to 319 with the average of 220 amino acids (Table 1). Nomenclature of putative MnLBD genes was carried out based on the scaffold orders and they were termed MnLBD1 to MnLBD31. The mulberry LBD genes were scattered over 28 scaffolds. Of them, MnLBD3, MnLBD4, and MnLBD5 were arrayed along the scaffold 40, while MnLBD13 and MnLBD14 were located closely on the scaffold 594. As shown in Table 1, the majority of MnLBD genes (74.2%) have one intron and six MnLBDs are intronless genes. Only two genes, MnLBD10 and MnLBD13 are intervened by two introns.

Phylogenetic distribution and gene structure of MnLBD genes

The protein sequences of all MnLBD genes were used to build a phylogenetic tree, in which 31 MnLBDs were separated into two classes, class I and class II (Fig. 1 and Supplementary material 2). Class I containing 26 proteins was further divided into five groups named class Ia to Ie. The gene structures of all MnLBD genes are illustrated in the right panel of Fig. 1. The data clearly showed that MnLBDs in class II have only one phase 1 intron. Most of the MnLBDs in class I also have a structure similar to the genes in the same subclass. For example, six genes in class Ib are intronless, seven of class Ic have one phase 0 intron, and three of class Id genes also have one phase 0 intron. However, there were two exceptions. MnLBD10 and MnLBD13, both of which are two-intron genes with atypical structures, are grouped in class Ia and class Ic, respectively.

Fig. 1

Phylogenetic analysis (left) and exon intron structures (right) of MnLBD genes. Numbers above or below branches of the tree indicate bootstrap values and the values below 50 are hidden. MnLBD genes are divided into two classes (Class I and Class II) in which Class I family was further divided into 5 groups and named from class Ia to Class Ie (left). Exons are shown by solid green bars and introns by the connecting lines. The numbers 0, 1, 2 represent the intron phase. The length of the genes can be estimated using the scale on the bottom (right).

As shown in Fig. 2, the results of the multiple sequence alignment indicated that a sequence with about 100 amino acids was conserved in all MnLBDs. For the class I MnLBDs, a string of blocks of C, GAS, and L-rich was recognized. Block C in MnLBDs can be summarized as: C-x(2)-C-x(6)-C-x(3)-C. Block GAS beginning with a F-x(2)-(V/A)-H motif and ending with a DP-(V/I)-YG motif. All class II MnLBDs have the conserved C-block similar to Class I and are absent in GAS-block and Leu-zipper like domain.

Fig. 2

LBD-domain alignment and corresponding logo of MnLBDs. Alignment of MnLBDs Class I genes was shown upside, and the conserved C block, GAS block and L-rich block are indicated by a red box. Alignment of MnLBDs Class II genes was shown below. The only conserved C block is represented by a red box. Below the alignment are the sequence logos. The overall height of each stack represents the degree of conservation at this position, while the height of the letters within each stack indicates the relative frequency of the corresponding amino acids.

Phylogenetic analysis of the LBD proteins

A. thaliana is a model plant species and the functions of some Arabidopsis LBD genes have been well-characterized, therefore, we constructed a phylogenetic tree with LBD protein sequences from A. thaliana and mulberry to provide insight into the functional prediction of mulberry LBDs. As shown in Fig. 3 (Supplementary material 3), all LBDs were separated into two classes, and most of them belonged to class I. There were 3 MnLBDs that strongly supported mulberry/Arabidopsis pairwise proteins with a bootstrap value of 99 shown in gray boxes on the phylogenetic tree, namely, MnLBD27/AtLBD06, MnLBD24/AtLBD21, and MnLBD20/AtLBD22. Furthermore, the phylogenetic relationship of MnLBD3/AtLBD6, MnLBD4/MnLBD5/AtLBD29/AtLBD17, MnLBD13/AtLBD19 and MnLBD14/AtLBD18 were close.

Fig. 3

Phylogenetic tree based on the Arabidopsis and mulberry LBD protein sequence. The bootstrap values are shown at the nodes and the values less than 50 are hidden. The AtLBDs and MnLBDs are marked with hollow and solid diamond respectively. The scale bar in the circle tree represents 0.5 substitutions per sequence position. The gray boxes indicate pairwise mulberry/Arabidopsis orthologous.

Expression analyses of putative MnLBDs

A heat map was created to check the expression profiles of various MnLBD in leaf, root, bark, bud, and flower. Based on this map, the 31 MnLBDs were classified into 5 groups. Group I consisted of 3 genes (MnLBD14, 18 and 22) which showed high transcript accumulation in leaves. Group II comprised of the genes MnLBD5, 15, 23, 25 and 29, which had a bark-bias expression. Thirteen genes (MnLBD3, 4, 7, 8, 9, 10, 11, 12, 13, 17, 19, 26 and 28) in group III were preferentially expressed in the root. In this group, it is worth mentioning that MnLBD13 was expressed not only in root, but also in the bark and flower. Five genes, MnLBD6, 20, 24, 30 and 31 in group IV, were expressed at relatively higher levels in the flower, and the expression of MnLBD1, 2, 16, 21 and 27 in Group V was detected mainly in bud (Fig. 4).

Fig. 4

Expression profiles of MnLBD genes. The clustering of genes was done by hierarchical clustering using average linkage clustering as rule with the default option after adjusting data in normalized genes/rows. Differences in gene expression are shown in color according to the scale. Different organs (root, bark, bud, flower and leaf) of mulberry were used for expression profiling, which are mentioned on top of each column listed. On the left side of expression map, clade names were given. Color bar at the top represents log2 expression values, wherein the green color represents low level expression, black shows medium level expression and red signifies a high level of expression. Five groups were divided according to the expression profiles.

Discussion

Our knowledge of plant LBD proteins has increased significantly since 2002. Shuai et al. identified a domain in 42 Arabidopsis proteins that are now referred to as LBD domain. The availability of sequenced plant genomes since its discovery has made it possible to isolate and study these genes. For example, through genome-wide analysis, 35 and 58 of LBD genes were identified in rice and apple, respectively (Yang et al., 2006, Wang et al., 2013). In the present study, it was revealed that the mulberry genome has 31 genes having LBD domain, in which 23 have a single intron interrupting the coding region while 6 are intronless. Two short genomic regions with clusters of MnLBD genes have been sequenced with 2–3 genes in a cluster. Understanding the consequences of gene expansion and diversification of the MnLBD genes is compelling. The development of various tissues in specific locations at specific time helps to determine the effect of diversification on the mulberry tree.

Sequence information and gene expressional data of the MnLBDs will facilitate future identification of candidate genes. Plant root system has an important role in both their response to soil conditions and tillage of the soil (Russell, 1977). Four genes in class Ia, MnLBD 3, 4, 8, and 13 are preferentially expressed in the mulberry root. Phylogenetic analysis revealed that, MnLBD3 was the most identical to Arabidopsis LBD16, which functions in lateral root development (Okushima et al., 2007). The other 3 genes, MnLBD4, MnLBD8 and MnLBD13 may also relate to lateral root development according to the expression profile and the phylogeny relationship with A. thaliana counterparts. Further study of these genes will contribute to a better understanding of the mechanism of mulberry in root development. In the same way, gene MnLBD22 was highly expressed in the leaf and placed in a branch with AtLBD36, which played an important role in leaf morphogeny (Nakazawa et al., 2003, Chalfun-Junior et al., 2005). These data may be used to hypothesize the role of MnLBD22 in mulberry leaf development.

On the other hand, mulberry has a variety of active secondary metabolites, such as flavonoids (Du et al., 2003), alkaloids (Asano et al., 2001), and terpenoids (Zhi-ming et al., 2012). However, the mechanism of mulberry secondary metabolism has not been well documented. It has been reported that the Arabidopsis gene AtLBD39 is involved in the biosynthesis of anthocyanins (Rubin et al., 2009). Gene MnLBD19 in class II is the closest homolog of AtLBD39 might implicate in the secondary metabolism of mulberry.

Conclusions

In the present study, thirty-one putative MnLBD genes were identified in the mulberry genome. Data on the expression of each of the Morus LBD genes coupled with sequence analysis provides valuable information for functional studies of mulberry LBD genes.

Conflict of interest statement

The authors declare that they have no competing interests.