Decipher the ancestry of the plant-specific LBD gene family
- Yimeng Kong†1,
- Peng Xu†2,
- Xinyun Jing1,
- Longxian Chen1,
- Laigeng Li2Email author and
- Xuan Li1Email author
© The Author(s). 2017
Published: 25 January 2017
Lateral Organ Boundaries Domain (LBD) genes arise from charophyte algae and evolve essential functions in land plants in regulating organ development and secondary metabolism. Although diverse plant species have been investigated to construct the phylogeny of LBD gene family, a detailed and reliable ancestry that characterizes their evolutionary patterns has not been revealed.
We develop an improved bioinformatic method that allows robust detection of 431 LBD genes in 11 high-quality land plant genomes. Phylogenetic analysis classifies the LBD genes into six subfamilies which support the existence of 7 ancient gene lineages. Phylogenetic relationship and gene collinearity are combined to retrace 11 ancestor genes for seed plants and 18 ancestor genes for angiosperms, which improves the resolution of LBD gene ancestry. The ancient gene lineages are strictly preserved in current plant genomes, including the previously controversial class IB gene in Selaginella moellendorphii, suggesting extreme reluctance of LBD genes to be lost during evolution. Meanwhile, whole-genome and dispersed gene duplications substantially expand LBD gene family in angiosperms, and elaborate functions of LBD genes through frequent expression pattern change and protein sequence variation.
Through phylogenetic and gene collinearity analyses, we retrace the landscape of LBD gene ancestry which lays foundation for elucidating evolutionary diversification of LBD genes in land plants.
Lateral Organ Boundaries (LOB) Domain (LBD) proteins define a group of plant-specific transcription factors that originated from charophyte algae [1, 2]. They share a characteristic LOB domain (also referred to as AS2 domain) with a conserved C-motif (CX2CX6CX3C), a Gly–Ala–Ser (GAS) block, and a leucine-zipper-like coiled-coil motif [3–5]. The C-motif is predicted to bind to DNA sequence, while the coiled-coil motif functions in mediating protein-protein interaction. In Arabidopsis, LBD genes were first identified for the specific expression at the base of lateral organs and the noticeable function in regulating leaf development [3, 4]. Subsequent studies showed diverse functions of LBD genes in regulating plant organ development and secondary metabolism: AtLBD6/AtAS2 regulates leaf formation ; AtLBD16, AtLBD18 and AtLBD29 control lateral root formation ; AtLBD27/SCP is required for microspore cell divisions ; AtLBD37, AtLBD38 and AtLBD39 are negative regulators of anthocyanin biosynthesis and N availability signals . It is frequently observed that LBD genes exhibit similar biological functions in different angiosperm species. For example, AtLBD16 orthologs control lateral root formation in Arabidopsis, rice and maize [6, 9, 10], and AtLBD6/AtAS2 orthologs repress meristematic cell formation and regulate abaxial-adaxial leaf polarity in Arabidopsis and maize [11–14]. The conserved roles of LBD gene orthologs suggest their functions may have been established prior to angiosperms emergence.
Extensive efforts have been exerted to classify LBD gene family in diverse plant species. In Arabidopsis, two major classes of LBD genes are traditionally classified according to the LOB domain structure [3, 4]. Class II proteins are distinctive to the class I due to the absence of the coiled-coiled motif. Further subdivisions of the two major classes reveal highly dynamic patterns in different species. In rice and maize, five subgroups are divided among class I LBD genes [15, 16]. In Arabidopsis and Malus, four and nine subgroups of class I LBD genes are classified respectively [5, 17]. The inconsistent subgroup number in these studies is likely caused by extensive duplications of LBD genes in angiosperms, and a single plant genome may not encompass their full diversity.
Recent studies performed more comprehensive analysis of phylogenetic distribution of LBD genes in multiple species spanning bryophytes, lycophytes and seed plants [1, 2]. Their results accordantly subdivide LBD genes into class IA, IB, IC1/ID, IC2, IE and class II, but there are still debates on whether class IB LBD genes are present in the genome of lycophytes [1, 2]. Moreover, angiosperm genomes retain large-scale collinear gene blocks which provides direct evidence to identify orthologous genes descendent from a common ancestor . But none of above studies investigates collinearity of LBD genes to infer their ancestors at each stage of land plant evolution. Therefore, a detailed and reliable ancestry that describes evolutionary history of LBD genes has not been revealed. Here we develop an improved method for LBD gene detection in 11 representative plant species. Through investigating gene collinearity and phylogenetic relationships, we present a detailed ancestry of LBD genes which characterizes their diversifying patterns in land plants.
Results and discussion
An improved method to identify LBD genes
Expansion patterns of LBD genes in angiosperms
Previous studies demonstrate genes descendent from a common ancestor often share chromosomal collinearity in angiosperms . Therefore, we analyze collinearity relationship of LBD genes to infer their inter-species orthology. Through iterative clustering of collinear LBD genes, we merge 133 LBD genes of dicots into 21 collinear groups and 92 LBD genes of monocots into 23 collinear groups (Fig. 2b and Additional file 2). The collinear groups contain 77% of total LBD genes in the analyzed angiosperms and each group represents a set of LBD gene orthologs originated from a common ancestor. Merging collinear groups into a higher hierarchy is not feasible because extensive genome fragmentation and rearrangement obscured syntenic blocks between dicots and monocots . Although the result identifies less than 23 members of LBD gene ancestors, additional phylogenetic information is needed to collapse the collinear groups of dicots and monocots to reflect a common angiosperm ancestor.
Retracing LBD gene ancestors in land plants
Gene collinearity and phylogenetic relationships are combined to retrace angiosperm ancestors of LBD genes. The ML tree categorizes 18 angiosperm gene clusters that include both dicot and monocot LBD genes (Fig. 4 and Additional file 3). Most of them are supported by inter-species gene collinearity except one cluster in class IE, which may be caused by massive gene duplications that likely obscure the syntenic block detection. Therefore, we propose that 18 LBD gene ancestors have been established prior to angiosperm emergence.
Reconstruct the ancestry of LBD gene family
We reconstruct evolutionary history of LBD genes with the deduced ancestor genes. In early land plants, 7 ancient lineages of LBD genes were established and remained in a stable amount until the divergence of seed plants (Fig. 4). Two rounds of gene duplication occurred before the emergence of seed plants and angiosperms, expanding LBD gene family to 11 and 18 members respectively. Further expansion of LBD genes in individual angiosperm species is highly associated with WGDs. The basal eudicot Aquilegia contains 26 LBD genes, while the amount could be increased to over 40 members in other eudicots which have undergone genome triplication or additional WGD events (Fig. 1) . The analyzed monocots have survived from two successive WGDs and contain more than 34 LBD genes in the genome . Noticeably, all of the major lineages of LBD genes could be detected in current angiosperm genomes, indicating they are extremely reluctant to be lost during evolution.
Diversification of LBD gene subfamily
To deepen annotation and classification of LBD gene family, we developed sequence profile features for each LBD gene class using whole protein sequences alignment. The result shows that, besides the well recognized LOB domains, different gene classes possess specific characteristic protein sequences (Additional file 5). The location of these characteristic sequences varies among different classes. They can lie immediately downstream the LOB domain (class IA), present at C-terminal regions (class IB and IC1/ID), or extend flanking the LOB domain (class IC2).
Class IA is composed of two ancient lineages, namely IA-1 and IA-2, which give rise to AtLOB and AtLBD6/AS2 in Arabidopsis respectively (Fig. 4). Consistent with the independent gene ancestry, AtLOB and AtLBD6/AS2 exhibit divergent biological functions even with highly similar amino acid compositions . Class IA-2 experienced a duplication event before angiosperm emergence and generates AtLBD6/AS2 lineage and AtLBD10 lineage. Sequence alignment identifies a “SKYQ” motif immediately downstream of the LOB domain in AtLBD6/AS2 orthologs (Additional file 7). In contrast, AtLBD10 orthologs contain a totally different sequence featured with “AAYIGP”. This phenomenon suggests protein sequence change is also accompanied with evolution of class IA LBD genes in angiosperms.
Class II subfamily contains two LBD gene ancestors before seed plant appearance. In Arabidopsis, AtLBD37, AtLBD38 and AtLBD39 are derived from a common ancestor (Fig. 4) and participate in a same biological process to repress anthocyanin biosynthesis and nitrogen responsive genes . Through sequence alignment, we identify a featured pattern of LxLxL motif in these proteins (Additional file 7) which is required to recruit TPL/TPR co-repressors and fulfill transcriptional repression activity . The LxLxL motif is also conserved in class II LBD genes of Physcomitrella, suggesting the ability to recruit TPL/TPR co-repressors has been acquired by early land plants.
In this study we present an improved method for LBD gene detection, and identify 431 LBD genes in 11 high-quality genomes of land plants. Through gene collinearity and phylogenetic analyses, we retrace 7 ancient LBD gene lineages in early land plants, which gave rise to 11 ancestor genes for seed plants and 18 ancestor genes for angiosperms through gene duplications. All of the ancient gene lineages are preserved by current genomes of land plant, including the previously controversial class IB gene in S. moellendorphii, suggesting LBD genes are extremely reluctant to be lost during evolution. On the other hand, whole-genome and dispersed gene duplications, accompanied with frequent protein sequence change and expression pattern alteration, account for the major expansions of LBD genes in angiosperms, which illustrates an important scheme for LBD gene family diversification.
LBD protein sequences are retrieved from public genome databases, including Aquilegia coerulea (JGI v1.1); Arabidopsis thaliana (TAIR 10); Eucalyptus grandis (JGI v2.0); Oryza sativa subsp. japonica (MSU v7.0); Picea abies (ConGenIE v1.0); Physcomitrella patens subsp. patens (JGI v3.0); Populus trichocarpa (JGI v3.0); Sorghum bicolor (JGI v2.1); Selaginella moellendorffii (JGI v1.0); Vitis vinifera (Genoscope 12X); Zea mays (MaizeSequence Release 6a). The LOB domain (designated as DUF260 in the PFAM database) is searched to identify putative LBD proteins. Hmmsearch (HMMer package version3.1b1) is used to search the PFAM profile hidden Markov model (pHMM) DUF260.hmm (http://pfam.xfam.org/)  against protein sequences from each genome. To ensure the searching reliability, domain hits beyond the gathering threshold (E-value 1e-5) and less than 80% of the coverage are filtered out before downstream analysis. We also remove redundant protein sequences which are alternatively spliced from the same locus. LBD genes in Arabidopsis and maize were named according to previous studies, while gene sequences from other species were renamed for simplicity (Additional file 1).
Gene duplication events and collinearity relationship analysis
MCscanX is used to detect gene duplication types and collineartiy relationships . For seven angiosperms (A. thaliana, E. grandis, P. trichocarpa, V. vinifera, O. sativa, S. bicolor and Z. mays), all annotated proteins in each genome were self-to-self compared by BLASTP (version 2.2.21) program with E-value 1e-10. The top 5 BLASTP hits of each gene were retained for downstream analysis of syntenic regions. The duplicate_gene_classifier program incorporated in MCscanX is used to identify different duplication types in a genome: whole genome duplication (collinear genes in syntenic blocks), tandem duplication (consecutive repeat genes), proximal duplication (genes spanning less than 20 genes in nearby chromosomal region) and dispersed duplication (other modes than whole genome, tandem and proximal duplications).
MCscanX is used to detect collinear blocks within dicot or monocot separately. Protein sequences of dicots (A. thaliana, E. grandis, P. trichocarpa and V. vinifera) or monocots (O. sativa, S. bicolor and Z. mays) were pooled independently to conduct self-self comparison by BLASTP (version 2.2.21) program with same parameters as described above (‘-e 1e-10 -b5 -v5 -m8’). PERL script detect_collinearity_within_gene_families.pl incorporated in MCscanX is used to detect collinearity within LBD gene family. LBD genes derived from collinear blocks are recursively merged into collinear groups using custom PERL script.
To generate phylogenetic tree of LBD genes in land plants, the protein sequences of LBD genes are aligned to PFAM profile hidden Markov models of the LOB domain (pHMM DUF260.hmm) using HMMalign (HMMer package version 3.1b1) . Terminal tails of non-aligned residues are trimmed using parameter ‘--trim’ and only unambiguous alignment of each sequence is subjected for subsequent phylogenetic analyses. The Jones, Taylor, and Thorton (JTT) model is selected as the best-fitting amino acid substitution model according to the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) scores estimated by ProtTest (v3.3) . The maximum likelihood (ML) analysis is performed by the program PhyML (version 3.1) using the JTT model of amino acid substitution, four gamma-distributed rate categories and the Shimodaira-Hasegawa-like approximate likelihood-ratio test (SH-aLRT) . Reliability of the internal branches is evaluated based on SH-aLRT supports. The tree is started from BIONJ tree and the topology of the tree is improved by subtree pruning and regrafting (SPR) method from 10 random starting trees. The output tree is visualized in the program Figtree (http://tree.bio.ed.ac.uk/software/figtree/).
To analyze class IA LBD genes, complete protein sequences were aligned with MUSCLE (v3.7) with default parameters . Multiple sequence alignments were trimmed by removing poorly aligned regions using TRIMAL (v1.4.rev15) with the option ‘-automated1’ . The ML analysis is performed by the program PhyML (version 3.1) using the JTT model of amino acid substitution, four gamma-distributed rate categories and SH-aLRT test. The tree is started from BIONJ tree and the topology of the tree is improved by SPR method from 10 random starting trees.
Sequence logos of LBD proteins
For each LBD class, MUSCLE (v3.7) was used to align the complete protein sequences with default parameters . Nucleotide conservation was hereafter analyzed and shown with WebLogo (v3.3) (http://weblogo.threeplusone.com/).
Selection pressure analysis for class IB LBD genes
Selection pressure analysis is measured by ω parameter in PAML (v4.6) package , which is the nonsynonymous/synonymous substitution rate ratio. Two hypotheses of variable selection pressure are modeled as H0 and H1. While the null model (H0) assigns only one ω for the whole tree, the branch model (H1) assigns two independent ω values for two branches. Codeml program is used to obtain the log likelihood by performing multiple analyses with a range of initial values for the ω parameter. Significant likelihood ratio tests (LRTs) is used to conduct the significance of difference between two models by chi2 program.
This article has been published as part of BMC Genomics Volume 18 Supplement 1, 2016: Proceedings of the 27th International Conference on Genome Informatics: genomics. The full contents of the supplement are available online at http://bmcgenomics.biomedcentral.com/articles/supplements/volume-18-supplement-1.
This work is supported in part by grants from the National Key Basic Research Program of China (2012CB316501, 2013CB127005 and 2012CB114502), the National Natural Science Foundation of China (31571310, 31401128, 31271409 and 31130012), Ministry of Agriculture of China (2016ZX08010-002) and special Fund for strategic pilot technology Chinese Academy of Sciences (XDA08020104). Publication costs for this article were funded by the National Key Basic Research Program in China (Nos. 2012CB316501) and the National Natural Science Foundation of China (31130012).
Availability of data and material
The data sets supporting the results of this article are included within the article and its additional file.
YK performed the data collection and analysis. PX designed the study and composed the manuscript. XJ and LC performed the realtime PCR analysis and participated in data analysis. XL and LL directed the research project and edited the manuscript. All the authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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