Identification and characterization of the Populus trichocarpa CLE family
BMC Genomics volume 17, Article number: 174 (2016)
The CLE (CLAVATA3/Endosperm Surrounding Region-related) gene family encodes small signaling peptides that are primarily involved in coordinating stem cell fate in different types of plant meristems. Their roles in vascular cambium have highlighted their potential function in wood formation. Apart from recent advances on identification and characterization of CLE genes, little is known about this gene family in a tree species.
Fifty PtCLE genes were identified from the Populus trichocarpa genome and were classified into four major groups based on sequence similarity. Analysis of the genomic organization of PtCLE genes indicates that genome duplication, as well as the diversity in the CLE motif, have contributed to the expansion of CLE gene family in poplar. A comparison with functionally characterized Arabidopsis CLE protein sequences showed that many PtCLE proteins are closely related to their predicted Arabidopsis counterparts. Particularly, PtCLE3, PtCLE12, PtCLE14 and PtCLE38 comprised an identical CLE motif to AtCLE41/TDIF, which is known as a regulator of vascular cambium homeostasis, strongly supporting the idea that similar signaling pathways exist in both species to regulate wood formation and secondary growth. Transcriptome profiling revealed that PtCLE genes generally were differentially expressed while some PtCLE genes exhibited tissue-specific expression patterns. Moreover, compared to their Arabidopsis counterparts, PtCLE genes showed either similar or distinct expression patterns, implying functional conservation in some cases and functional divergence in others.
Our study provides a genome-wide analysis of the CLE gene family in poplar, and highlights the potential roles of key PtCLE genes in the regulation of secondary growth and wood formation. The comparative analysis revealed that functional conservation may exist between PtCLEs and their AtCLE orthologues, which was further supported by transcriptomic analysis. Transcriptional profiling provided further insights into possible functional divergence, evidenced by differential expression patterns of various PtCLE genes.
Small regulatory peptides, a growing class of signaling molecules mediating cell-cell communication, are essential for plant growth, development and responses to environmental stimuli [1–6]. The CLE (CLAVATA3/Endosperm Surrounding Region-related) peptide family is one of the well-studied peptide families in plants. The CLE genes have been found in many plant species and some plant parasitic nematodes, while the functions of most CLE genes are still unknown [2, 3, 7–13]. However, accumulated data have revealed that CLE genes played vital roles in stem cell homeostasis of different types of plant meristems including the SAM (Shoot Apical Meristem; AtCLV3), the RAM (Root Apical Meristem; AtCLE40, AtCLE19 and AtCLE22), the vascular meristem (AtCLE41/TDIF) and the root nodule meristems (LjCLE-RS1/2; MtCLE12/13; GmRIC1/2) [14–27].
Other than their roles in stem cell homoeostasis, CLE genes have been found to participate in a range of biological processes [2–6]. AtCLE1, AtCLE3, AtCLE4, and AtCLE7, for example, were predominantly expressed in the Arabidopsis root pericycle, and their expressions were induced under nitrogen-deficient conditions . Over-expression of AtCLE1, AtCLE3, AtCLE4, and AtCLE7 repressed the emergence and growth of lateral roots, which required CLV1, suggesting that CLV1 mediated a nitrogen-responsive CLE peptide signaling pathway that negatively regulated later root development under nitrogen deficiency . AtCLE8 is specifically expressed in the endosperm and young embryos . The mutation of AtCLE8 caused smaller and defective seeds/embryos, while ectopic expression of the AtCLE8 gene resulted in larger seeds/embryos, indicating that AtCLE8 played crucial roles in embryogenesis and endosperm development . Overexpression of HgCLE1, a CLE-like nematode gene, resulted in a wus-like phenotype and a short-root phenotype. Consistently, overexpression of HgCLE1 rescued the clv3-1 mutant phenotype . Further studies have shown that multiple receptors, including CLV1, RPK2, CRN/SOL2 and CLV2, are required for the successful nematode infection of Arabidopsis roots [31, 32].
It has been shown that a number of CLE genes, including AtCLE6, AtCLE10, AtCLE19, AtCLE41/TDIF and AtCLE44, played roles in vascular development [5, 33, 34]. In particular, exogenous application of AtCLE41/TDIF peptides inhibited xylem vessel differentiation, but had no effect on the SAM and/or RAM development. Consistently, over-expression of AtCLE41/TDIF resulted in a xylem vessel strand-discontinuous phenotype in a PXY/TDR-dependent manner [18, 22, 23]. Intriguingly, both over-expression and exogenous peptide application promoted cambial cell proliferation [19, 23]. In combination, the data suggested that AtCLE41/TDIF promoted the proliferation of vascular cambium cells while preventing them from differentiating into xylem through the TDR/PXY receptor [19, 22, 23]. Recently, it has been suggested that the AtCLE41/TDIF-PXY/TDR signaling module is evolutionarily conserved on regulating the secondary growth in poplar tree species . By tissue-specific over-expression of PttCLE41 and PttPXY genes, Etchells and colleagues (2015) generated poplar trees that exhibited enhanced growth and increased wood formation .
Poplar has been proposed as a model plant in understanding the molecular basis of tree growth and development, particularly the formation of wood which is commercially used for manufacturing, such as fuel and construction materials . However, little is known about CLE genes in this economically important tree species. As the conservation of their fundamental roles in the regulation of maintenance and differentiation of meristematic tissues, particularly the cambium, as well as other cellular processes, it is of great interest to study the CLE gene family in poplar, with an focus on CLE genes exhibiting expression in vascular tissues which might be important for wood formation. With the availability of the genome sequence of poplar (Populus trichocarpa), we carried out a genome-wide analysis to identify CLE genes as a first step to gain insights into their potential roles in various aspects of poplar growth and development, enabling a better understanding of the CLE gene family in a tree species.
Results and discussion
Identification and annotation of the CLE family in Populus trichocarpa
Systematic TBLASTN and BLASTP analyses were performed using previously reported CLE proteins and CLE motifs from various plant species as queries searching against the Populus trichocarpa genome (http://www.phytozome.net/). The retrieved candidate genes were then filtered for proteins with an N-terminal signal peptide and a C-terminal conserved CLE motif . The analysis was iterated until no new CLE candidate was identified. As a result, a total of 50 PtCLE (Populus trichocarpa CLE) genes were identified (Table 1). Twenty-six PtCLE genes were reported previously , thus our current work identified 24 additional PtCLE members (Table 1).
Similar to Arabidopsis CLE proteins, PtCLEs displayed few sequence features with each other, apart from the secretion signals and the CLE motifs (Fig. 1; Additional files 1, 2, 3, 4, 5, 6 and 7). In line with the AtCLE members, the presence and location of putative N-terminal signal peptide cleavage sites were predicted in each PtCLE (Fig. 1; Additional file 1). It has been shown that deletion of the putative CLE signal peptide inactivated the CLE protein activity in vivo, suggesting that the signal peptide is essential for in vivo functions of CLE peptides .
The CLE proteins contain one or more C-terminal conserved CLE motif(s), which was reported to be a 12–13 amino acid hydroxyprolinated, triarabinosylated peptide, and was the functional domain of CLE proteins [38, 39]. MEME (Multiple Expectation Maximization for Motif Elicitation) was employed to investigate the presence and distribution of CLE motifs in all PtCLE proteins. Only one single CLE motif was found to be present across all PtCLEs (Fig. 1; Table 1; Additional files 1, 2, 3, 4, 5, 6 and 7). The presence of multiple CLE domains was not observed in any of the PtCLE proteins although CLE proteins containing multiple CLE domains have been reported previously (Table 1; Fig. 1; Additional files 1, 2, 3, 4, 5, 6 and 7; [10, 11]).
The CLE motif, a segment that contains the mature CLE peptide sequence, is highly conserved across all CLE proteins [37, 40]. As expected, the consensus sequences of the CLE motif between AtCLE and PtCLE are highly conserved (Fig. 1b-c; Additional file 3; Additional file 5), suggesting functional conservation between PtCLEs and AtCLEs. Similar to AtCLEs, residues R2, P5, G7, P8, P10 and H12 of the CLE motifs in PtCLEs are highly conserved (Fig. 1b-c). Only moderate conservation was observed for amino acids (V/S)4, (N/D)9 and (N/H)13, although a similar probability of occurrence presented in both AtCLEs and PtCLEs (Fig. 1b-c; Additional file 3; Additional file 5). These conserved residues might provide a framework for the physical binding with their presumed receptors. Studies have been reported that residues D, H, G, P5, R and P10 of the CLE domain were critical for proper AtCLV3 function in SAM as evidenced by Ala-substitutions . In addition, residues in the flanking sequences and the hydroxylation/arabinosylation modifications of residue P8 are also critical to the AtCLV3 function [42–44]. Furthermore, a Gly-to-Thr substitution in the CLE motifs resulted in a strong dominant-negative effect . However, to what extent the conservation of these residues in the CLE motif across poplar and Arabidopsis could reflect their functional relevance awaits further investigation. Furthermore, the CLE motif exhibited residue divergence at positions 1, 3, 6 and 11 (Fig. 1b-c, Additional file 3; Additional file 5), which may provide the basis for distinct functions of the individual PtCLEs and/or the specificity of the putative receptor(s) binding.
Four or five residues proximal to the CLE motif at the N-terminus are required for proper endoproteolytic processing and optimal function in stem cell regulation [44, 45]. A comparison of the six residues (6-AA) proximally adjacent to the CLE motifs revealed high divergence across all PtCLEs, but a degree of residue conservation was found for multiple PtCLEs (Additional files 6 and 7). A Lys residue is presented before the conserved Arg residue of the 12-AA CLE motif in many PtCLEs (Additional files 6 and 7). This may suggest that the importance of this residue for endopeptidase recognition, which has been shown in the case of AtCLV3 and AtCLE1 . Additionally, 17 out of 50 PtCLEs carried an Arg residue immediately following the CLE motif at the C-terminus, indicating a possible decrease of peptide activity as has been reported previously .
The PtCLE proteins are classified into four major distinct groups
Although PtCLE proteins shared little sequence similarity, the CLE motifs were well conserved. Therefore, all the CLE motif sequences, as well as the full length proteins, were used as the basis to build phylogenetic trees separately. Phylogenetic analyses using several methods supported the classification of PtCLE proteins into four major groups (Fig. 2a; Additional file 8). The CLE motifs of the four groups were aligned, which resulted in consensus sequences supporting for classification of these four groups (Fig. 2b).
The consensus sequences of the CLE motifs in all groups (positions 7–13) were highly conserved with five residues that were almost invariant, except for position 11 of Group II and position 12 of Group IV (Fig. 2b). However, residue divergence across the first six N-terminal residues of the CLE motif was observed in all groups, especially in Group IV, in which high variance was observed (Fig. 2b). The CLE motifs of Groups I, II and III lacked the conservation of the Ser residue at position 6, which was invariant in Group IV (Fig. 2b). The Lys residue at position 1 of Group I was highly conserved, whereas the residue at the same position of other groups was rather variable (Fig. 2b). Group II contained a group-specific Ser residue at position 4, which may be largely responsible for its separation into a distinct group (Fig. 2b). However, whether the conserved residues and/or distinct group-specific residues contribute to CLE functionalities requires biological validation.
Previously, CLE proteins identified from various plant species were categorized into thirteen groups . A closer examination of the CLE consensus sequences revealed that Groups I, II, III and IV of PtCLEs corresponded to Groups 7, 2, 9 and 5 presented in , respectively. The comparison indicated a similar signature of the CLE motifs in both classifications. It was reported that Arabidopsis CLE was classified into four functional groups based on the effects of peptide treatment on plants , which was well correlated with the phylogenetic analysis of AtCLEs [23, 48, 49]. The classification presented in  contained at least one functional CLE in each group, which helped to understand the possible function(s) of individual PtCLE group. Nevertheless, the correlation of phylogenetic analyses between ours and  implied strong functional similarities between interspecies orthologs as validated by many functional characterized CLE genes from Arabidopsis, rice, Medicago, Lotus japonicus and soybean [2, 3]. For instance, Group IV members, which correspond to the Group 5 as classified in , were predicted to confer similar phenotypic effects on vascular development in poplar to those observed in Arabidopsis [11, 18, 22, 23]. However, determining whether these predicted gene functions are evolutionarily conserved requires further biological investigation.
Genomic organization of PtCLE genes
Similar to AtCLE genes, PtCLE genes often lacked introns. Only thirteen PtCLE genes contained intron(s), seven of which contained one intron and six of which contained two introns (Fig. 3a). PtCLE genes scattered over on different chromosomes although some clustering can be observed (Fig. 3b). Furthermore, some of the PtCLE genes were found to be located adjacently to each other (Table 1; Fig. 3b). For instance, PtCLE7 and PtCLE8, PtCLE36 and PtCLE37, PtCLE40 and PtCLE41, PtCLE45 and PtCLE46 were located in tandem on chromosomes 1, 9 12 and 15, respectively. Additionally, PtCLE48, PtCLE49 and PtCLE50 were organized sequentially in tandem on chromosome 19 (Table 1; Fig. 3b). However, sequence comparison within those tandem pairs showed low sequence similarity, and the CLE motifs were not totally identical, implying that these genes might not arise from recent tandem duplication events (Table 1; Fig. 3b; Additional files 2 and 3). These observations may indicate, in some cases, that diversity in the CLE motifs was favored during evolution which may give rise to distinct roles of PtCLEs and expansion of the PtCLE gene family.
Interestingly, a number of PtCLE genes located on different chromosomes encoded identical, or nearly identical CLE motifs, suggesting that these PtCLE genes were possibly duplicated genes arising from segmental duplication events (Additional file 9). For instance, the CLE motifs of the positionally adjacent pairs PtCLE7/PtCLE8 were almost identical to that of PtCLE36/PtCLE37, while those of PtCLE40/PtCLE41 were nearly identical to that of PtCLE45/PtCLE46 (Table 1; Fig. 3; Additional files 2 and 3). Moreover, PtCLE3, 12, 14 and 38 comprised identical CLE motifs, while PtCLE21, 31 and 39 shared the same CLE motifs (Table 1; Additional file 9). A set of five pairs, PtCLE7/PtCLE36, PtCLE18/PtCLE27, PtCLE20/PtCLE32, PtCLE22/PtCLE30 and PtCLE28/PtCLE47, carried identical CLE motifs within pairs (Table 1; Additional file 9). These results suggested that genome-scale duplication of PtCLE genes occurred in different regions of poplar chromosomes. In tomato, neighboring SlCLE genes, sharing no significant similarity within pairs, were found on different chromosomes, suggesting that these neighboring SlCLE were not likely to arise through tandem duplication . However, it was observed that many AtCLE gene pairs, e.g., AtCLE9/AtCLE10 and AtCLE5/AtCLE6, may have arisen through duplication. Additionally, many AtCLE genes were found in regions of the genome that were rich in repetitive sequences . These results suggested that rearrangement and gene duplication were plausible mechanisms for the expansion of the AtCLE gene family . Therefore, like AtCLEs, genome duplication and reshuffling contributed to the expansion of PtCLE gene family . Moreover, unlike Arabidopsis, the subsequent diversity in the CLE motifs of PtCLEs also has driven the expansion of this family.
Probing the roles of PtCLE genes by phylogenetic analyses and expression profiles between Arabidopsis and poplar
As the first attempt to investigate potential role(s) of PtCLEs, the phylogenetic relationships between AtCLEs and PtCLEs were analyzed (Additional files 10 and 11). The phylogenetic analysis classified the PtCLEs and AtCLEs into several clades with varying degrees of phylogenetic distance based on the conserved CLE motifs that were used to construct the phylogenetic tree (Fig. 4; Additional file 10). Although the phylogenetic tree is based on the CLE motif alone, the clades defined by this tree correlated very well with phylogenetic relationship defined using full-length CLE proteins (Additional file 11).
Overall, our analysis indicated that the PtCLE proteins were quite closely related to their predicted Arabidopsis counterparts, which allowed interspecies identification of putative functional orthologs (Fig. 4; Additional files 10 and 11). Some clades segregated AtCLE and PtCLE proteins, whereas other clades contained CLE proteins of both species (Additional files 10 and 11). Each of these clades contained at least one functionally characterized member, allowing us to infer possible functions for the PtCLEs in the same clade (Additional files 10 and 11). Thus, the potential function of PtCLEs in each clade was predicted using functionally characterized AtCLEs [1–6, 49, 50]. As aforementioned, many PtCLE proteins contained perfectly matched CLE motifs (Additional file 9). Particularly, some PtCLE proteins comprised CLE motifs that matched completely with the CLE motifs of AtCLE proteins (Fig. 4; Additional file 9). It is presumed that PtCLEs with identical CLE motifs or PtCLEs carrying the same CLE motifs as that of AtCLEs might share similar protein functions [37, 40]. In addition to the CLE motif, the expression domain of CLE genes is also important for their functional specificities as have been shown that many AtCLE proteins acted interchangeably when ectopically expressed [37, 40, 45, 49, 51]. Therefore, we assessed potential roles of PtCLE genes using a combination of phylogenetic analyses and available transcriptomic data.
The roles of AtCLV3, AtCLE1/AtCLE3/AtCLE4, AtCLE9/AtCLE10 and AtCLE41/TDIF have been functionally characterized previously [14, 15, 18, 19, 22, 23, 28, 33, 52]. We thus further identified PtCLEs sharing identical or nearly identical CLE motifs with those well-studied AtCLEs. Three PtCLEs (PtCLE4, PtCLE17 and PtCLE25) were grouped together with AtCLV3, of which PtCLE4 and PtCLE17 shared a nearly identical CLE motif with AtCLV3 (Fig. 4a; Additional files 10 and 11). AtCLV3, perceived by various parallel receptor complexes, restricted expression of the stem cell-promoting transcription factor WUS, which in turn activated AtCLV3 expression, thus forming a negative feedback loop that maintained a balanced stem cell population [14, 15, 53–56]. Indeed, PtCLE4 showed a highest expression level in shoot apex and a moderate expression level in shoot and leaf primordia of all materials tested, strongly supporting a possible role for PtCLE4, similar to AtCLV3, in regulating poplar shoot development (Fig. 5; Additional file 12). However, we cannot exclude the possibility that PtCLE17 and PtCLE25 also play roles in shoots.
AtCLE1, AtCLE3 and AtCLE4 repressed the lateral root development in a CLV1-dependent manner in Arabidopsis . Two PtCLEs, PtCLE7 and PtCLE36, shared an identical CLE motif as that of AtCLE1/AtCLE3/AtCLE4 (Fig. 4b). PtCLE36 was found to predominantly expressed in the xylem (Fig. 5; Additional file 12), unlike what has been observed for AtCLE3 , suggesting a different role of PtCLE36. However, it still will be of great interest, as the first step, to investigate whether the expression of PtCLE7 and PtCLE36 are induced under nitrogen deficient conditions.
AtCLE9/AtCLE10 inhibited protoxylem vessel formation via CLV2 by repressing the expression of ARR5 and ARR6 in Arabidopsis roots . A pair of PtCLEs (PtCLE20 and PtCLE32) comprised the same CLE motif as that of AtCLE9/AtCLE10 (Fig. 4c). Similar to AtCLE10, PtCLE20 was highly expressed in vascular tissues (Fig. 5; Additional file 12). Numerous CLV2-like proteins have been mined from poplar , which favors the idea that a similar AtCLE9/AtCLE10-CLV2 signaling pathway regulates root vascular development in poplar as well.
A subclade of four PtCLEs (PtCLE3/PtCLE12/PtCLE14/PtCLE38) grouped together with AtCLE41/TDIF, sharing an identical CLE motif (Table 1; Fig. 4d; Additional file 3; Additional file 9), which strongly supported a conserved role of these peptides in the regulation of vascular cambium homeostasis in poplar and Arabidopsis. Intriguingly, in all materials tested, PtCLE12 had the highest expression level in phloem, and was almost absent from xylem (Fig. 5; Additional file 12). This expression pattern was similar to that of its putative Arabidopsis counterpart AtCLE41/TDIF . PtCLE3, another PtCLE gene encoding an identical CLE motif with that of AtCLE41/TDIF, was highly expressed in cambium and moderately expressed in phloem, which may suggest a broader role for PtCLE3 in poplar (Fig. 5; Additional file 12). In Arabidopsis, the plasma membrane-bound receptor PXY/TDR perceived the AtCLE41/TDIF to promote the (pro-)cambial proliferation by regulating WOX4 expression, and to suppress (pro-)cambial cell differentiation into xylem cells [22, 23]. Recently, Etchells et al.  showed that tissue-specific expression of PttPXY and PttCLE41 produced transgenic trees with increased wood production and a larger biomass. Notably, PttCLE41 was the same CLE protein as PtCLE38 identified in this study (Table 1; Fig. 4f). Altogether, it seems possible that PtCLE12 also plays a similar role in the regulation of cambium development and wood formation. However, we cannot exclude the possibility that PtCLE14 carrying the same CLE motif as AtCLE41/TDIF, is also involved in (pro-)cambium stem cell homoeostasis (Fig. 5; Additional file 12). Taken all together, this pointed to the existence of a similar AtCLE41/TDIF-TDR/PXY module in regulating secondary growth in trees. However, whether the other three PtCLE proteins (PtCLE3/PtCLE12/PtCLE14), which contained an identical CLE motif as that of PttCLE41/PtCLE38, sharing a similar function remained unknown. Four hundred receptor-like kinases (RLKs) and eighteen WUS-related proteins have been identified in poplar, which supports the idea that the existence of multiple CLE-RLK-WOX signaling pathways [58, 59].
In summary, we grouped and compared the PtCLE proteins with their most closely-related AtCLE proteins to assess their potential roles based on functional studies [2–4, 6, 35, 49, 50]. The study indicated that PtCLE proteins are generally closely related to their predicted Arabidopsis counterparts. Intriguingly, many PtCLE proteins comprised exactly the same CLE motifs as that of their Arabidopsis counterparts, strongly suggesting functional conservation between specific AtCLEs and PtCLEs. However, It is also possible that those PtCLEs carrying identical CLE motifs play distinct roles in planta which could be achieved via tissue-specific expression pattern. Additionally, a few sets of PtCLEs shared an identical or nearly identical CLE motif, whereas no closely-related AtCLEs could be identified in the phylogenetic clades (Additional files 9, 10 and 11), raising the possibility that these PtCLEs may have unique functions in woody trees. It is of great interest to examine whether those PtCLEs possessing similar CLE motifs are functionally redundant as what has been observed in the AtCLE gene family [17, 26]. Nevertheless, it is important to assess to what extent these observations are supported by biological validation.
Uncovering putative functions of PtCLE genes in shoot and vascular development
Previous studies have demonstrated that CLE peptides played various roles in plant growth and development [1–6]. To deepen our understanding of the potential functions of PtCLE proteins, in silico expression data for 30 out of 50 PtCLE genes were obtained from different Populus species other than P. trichocarpa for further analysis (Additional file 13). A total of six developmental microarray sets including samples derived from various organs and tissues were retrieved and normalized for further study with an emphasis on shoot organogenesis and vascular development (Additional file 13).
PtCLE genes generally exhibited differential expression patterns in the materials tested (Fig. 5; Additional files 12 and 13), similar to what was observed for the expression of AtCLEs . Other than PtCLE4, PtCLE11, PtCLE22, PtCLE29, and PtCLE45 are also highly expressed in shoot apex and/or shoot and leaf primordia (Fig. 5; Additional file 12). Among these, PtCLE22 and PtCLE29 showed consistent expression patterns in two tested samples. PtCLE45 is limited to the shoot apex, whereas PtCLE11 expression is relatively restricted to the shoot and leaf primordia, indicating a spatially and temporally expression fashion (Fig. 5; Additional file 12). However, whether any of these PtCLE proteins are involved in controlling the stem cell homoeostasis in shoot apex or in primordia remained to be proven.
The (pro-)cambium, a stem-cell tissue, gives rise to the phloem and xylem which perform essential roles in transportation of water, mineral nutrients and signaling molecules . In Arabidopsis, the AtCLE41/TDIP-TDR/PXY-WOX4 signaling module plays an important role in (pro-)cambium proliferation and differentiation [19, 22, 23]. Additionally, a number of AtCLEs are shown to control vascular development, which assigned CLEs as central players mediating cell-cell communication in plant vascular development . In our analysis, we found that a number of PtCLE genes are predominantly expressed in various vascular tissues, except the aforementioned PtCLE3 and PtCLE12 (Fig. 5; Additional file 12). PtCLE5, PtCLE26 and PtCLE34 are expressed at the highest level in cambium and xylem, while PtCLE10/PtCLE13/PtCLE21/PtCLE27/PtCLE35/PtCLE36 exhibited a peak expression level in xylem. PtCLE20 and PtCLE50 are predominantly expressed in the cambium (Fig. 5; Additional file 12). The expression of PtCLE24, PtCLE28 and PtCLE39 is mainly detected in the phloem (Fig. 5; Additional file 12). The transcriptional activities of the remaining PtCLE genes are highly dynamic (Fig. 5; Additional file 12).
Interestingly, we found that PtCLE gene pairs encoding identical CLE motifs, including PtCLE3/PtCLE12, PtCLE21/PtCLE39, and PtCLE28/PtCLE47, exhibited both overlapping and distinct expression patterns with respect to different tissues (Fig. 5; Additional file 12). This points to functional divergence of these PtCLE genes in planta despite that they share the same CLE motif. We further investigated whether expression trends are similar between AtCLE genes and their putative poplar orthologues. In addition to previously high-resolution expression data for the entire Arabidopsis A-type CLE genes , we compiled and visualized the expression profile of AtCLE genes in selected tissues by e-Northerns browser of BAR (Additional file 14; ). In silico expression data for 14 out of 32 AtCLE genes were available. In the case of AtCLE46, it was highly expressed in meristematic tissues and xylem-rich samples (Additional file 14). A similar expression trend was observed for its putative poplar orthologues PtCLE5 and PtCLE26, both of which exhibited significant expression levels in cambium and developing-/differentiating-xylem (Fig. 5; Additional file 12). However, only some CLE genes of Arabidopsis and poplar are presented in the microarrays, making it difficult for in-depth investigation. Nevertheless, it is also likely that other PtCLE genes which are not available on the microarrays show significant expression in some tissues. Thus we analyzed the available EST sequences and RNA-seq data to explore the expression of the PtCLE genes that are not presented in the microarray. The corresponding ESTs and RNA-seq reads were extracted from public databases, demonstrating that these PtCLE genes were transcribed based on the numbers of ESTs detected and the FPKM (the number of fragments per kilobase of exon per million fragments mapped) values for RNA-seq data (Additional file 15). In several cases, there are no matched EST(s) were identified in P. trichocarpa, but matched EST(s) from sibling species or high FPKM value could be detected (Additional file 15). The matched ESTs varied in numbers, suggesting that they are expressed differentially or the ones with few ESTs are probably expressed at low level or restrict to particular tissues or developmental stages. Altogether, our data indicated a complicated expression profile amongst the PtCLE genes, which is well correlated with their diverse roles in poplar growth and development.
The CLE genes are well known for their roles in coordinating stem cell fate in different types of plant meristems including the vascular cambium, which is the most notable growth characteristic in tree species. In this study, the CLE gene family in P. trichocarpa, a tree species with extensive wood formation, was identified and classified into four major groups based on sequence similarity. The potential roles of PtCLE genes, with an emphasis on shoot organogenesis, secondary growth and wood formation, were analyzed by comparative studies and transcriptional profiling. A number of PtCLE proteins and their putative Arabidopsis orthologues were identified based on identical or nearly identical CLE motifs and comparable tissue expression expression patterns, pointing to possible functional conservation of these CLE proteins. Conversely, some PtCLE genes appeared to be regulated in completely different ways from their Arabidopsis counterparts, which may provide insights into the functional divergence of CLE signaling in tress species. The comparative studies further indicated close parallel regulation of AtCLEs and PtCLEs orthologues, which highlighted potential strategies such as manipulation of key plant peptide signaling molecules for higher yields and more sustainable wood sources.
Identification of PtCLE proteins and protein features analysis
All known CLE proteins were retrieved and used as queries to perform the BLASTP and TBLASTN programs searching against the Populus trichocarpa genome sequence (http://www.phytozome.net; ). Each identified hit subsequently was used as a new query to conduct a BLASTP search querying against the poplar assembly genomic sequence (Version 2.2) to avoid any missed PtCLE protein. The searches were run repeatedly until no new candidates were found.
SignalP (http://www.cbs.dtu.dk/services/SignalP), Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme.nbcr.net/meme/cgi-bin/meme.cgi) , and Weblogo (http://weblogo.berkeley.edu/logo.cgi)  were used for domain predictions and determination of domain features. SignalP was run for determining the signal peptides using both neural network (NN) and hidden Markov model (HMM) modes. In the cases that SignalP yielded low scores, the TargetP (http://www.cbs.dtu.dk/services/TargetP), iPSORT (http://ipsort.hgc.jp) and SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP-2.0) were used to identify signal sequences.
Genomic organization analysis
The exon/intron boundaries of each PtCLE genes were investigated using gene structure display server (http://gsds.cbi.pku.edu.cn)  and refined manually with expression data of EST sequences and cDNA sequences that were deposited in Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ptrichocarpa). The chromosomal locations of PtCLE genes were determined using PopGenIE (http://popgenie.org/gp) .
Alignment and phylogenetic analysis
Multiple alignments were performed using ClustalX , then refined and displayed using GeneDoc (http://www.psc.edu/biomed/genedoc). Phylogenetic trees were constructed by MEGA5 using either the conserved CLE motifs or full-length CLE proteins . Bootstrap analysis was conducted with 1000 replicates to verify the significance of nodes.
Gene expression analysis
Microarray data were obtained from the Gene Expression Omnibus database (GEO) at NCBI website. As a result, six developmental microarray datasets were collected as shown in Additional file 13. The downloaded raw CEL files were analyzed using the Affy package in R language , followed by the background correction and microarray expression normalization using the RMA method . Differential gene expression was determined according to , which was followed by a multiple testing correction . Heatmaps were generated based on the expression profiles, in which cluster of PtCLE proteins were determined as well. The EST (Expressed Sequence Tags) sequences and RNA-seq data were obtained from Phytozome. Transcript abundances based on RNA-Seq data in mixed tissues were calculated as numbers of fragments per kilobase of exon in a gene per million fragments mapped (FPKM).
Availability of supporting data
Phylogenetic data have been deposited to TreeBase and are accessible via the URL: http://purl.org/phylo/treebase/phylows/study/TB2:S18866. Additional supporting data are included as additional files.
Wang G, Zhang G, Wu M. CLE peptide signaling and crosstalk with phytohormones and environmental stimuli. Front Plant Sci. 2016;6:1211. doi:10.3389/fpls.2015.01211.
Betsuyaku S, Sawa S, Yamada M. The function of CLE peptides in plant development and plant-microbe interactions. The Arabidopsis Book. 2011;9:e0149. doi:10.1199/tab.0149.
Murphy E, Smith S, De Smet I. Small signaling peptides in Arabidopsis development: how cells communicate over a short distance. Plant Cell. 2012;24(8):3198–217.
Miyawaki K, Tabata R, Sawa S. Evolutionarily conserved CLE peptide signaling in plant development, symbiosis, and parasitism. Curr Opin Plant Biol. 2013;16(5):598–606.
Qiang Y, Wu J, Han H, Wang G. CLE peptides in vascular development. J Integr Plant Biol. 2013;55(4):389–94.
Endo S, Betsuyaku S, Fukuda H. Endogenous peptide ligand receptor system for diverse signaling networks in plant. Curr Opin Plant Biol. 2014;21:140–6.
Mitchum MG, Wang X, Davis EL. Diverse and conserved roles of CLE peptides. Curr Opin Plant Biol. 2008;11:75–81.
Mitchum MG, Wang X, Wang J, Davis EL. Role of nematode peptides and other small molecules in plant parasitism. Annu Rev Phytopathol. 2012;50:175–95.
Cock JM, McCormick S. A large family of genes that share homology with CLAVATA3. Plant Physiol. 2001;126(3):939–42.
Sawa S, Kinoshita A, Betsuyaku S, Fukuda H. A large family of genes that share homology with CLE domain in Arabidopsis and rice. Plant Signal Behav. 2008;3(5):337–9.
Oelkers K, Goffard N, Weiller GF, Gresshoff PM, Mathesius U, Frickey T. Bioinformatic analysis of the CLE signaling peptide family. BMC Plant Biol. 2008;8:1.
Strabala TJ, Phillips L, West M, Stanbra L. Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and CLE-LIKE signal peptide genes in the Pinophyta. BMC Plant Biol. 2014;14:47.
Zhang Y, Yang S, Song Y, Wang J. Genome-wide characterization, expression and functional analysis of CLV3/ESR gene family in tomato. BMC Genomics. 2014;15:827.
Fletcher JC, Brand U, Running MP, Simon R, Meyerowitz EM. Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristem. Science. 1999;283(5409):1911–4.
Rojo E, Sharma VK, Kovaleva V, Raikhel NV, Fletcher JC. CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway. Plant Cell. 2002;14(5):969–77.
Casamitjana-Martinez E, Hofhuis HF, Xu J, Liu CM, Heidstra R, Scheres B. Root-specific CLE19 overexpression and sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance. Curr Biol. 2003;13(16):1435–41.
Fiers M, Hause G, Boutilier K, Casamitjana-Martinez E, Wijers D, Offringa R, et al. Mis-expression of the CLV3/ESR-like gene CLE19 in Arabidopsis leads to a consumption of root meristem. Gene. 2004;327(1):37–49.
Ito Y, Nakanomyo I, Motose H, Iwamoto K, Sawa S, Dohmae N, et al. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science. 2006;313(5788):842–5.
Hirakawa Y, Shinohara H, Kondo Y, Inoue A, Nakanomyo I, Ogawa M, et al. Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system. Proc Natl Acad Sci U S A. 2008;105(39):15208–13.
Okamoto S, Ohnishi E, Sato S, Takahashi H, Nakazono M, Tabata S, et al. Nod factor/nitrate-induced CLE genes that drive HAR1-mediated systemic regulation nodulation. Plant Cell Physiol. 2009;50(1):67–77.
Stahl Y, Wink RH, Ingram GC, Simon R. A signaling module controlling the stem cell niche in Arabidopsis root meristems. Curr Biol. 2009;19(11):909–14.
Etchells JP, Turner SR. The PXY-CLE41 receptor ligand pair defines a multifunctional pathway that controls the rate and orientation of vascular cell division. Development. 2010;137(5):767–74.
Hirakawa Y, Kondo Y, Fukuda H. Regulation of vascular development by CLE peptide-receptor systems. J Integr Plant Biol. 2010;52(1):8–16.
Mortier V, Den Herder G, Whitford R, Van de Velde W, Rombauts S, D’Haeseleer K, et al. CLE peptides control Medicago truncatula nodulation locally and systemically. Plant Physiol. 2010;153(1):222–37.
Lim CW, Lee YW, Hwang CH. Soybean nodule-enhanced CLE peptides in roots act as signals in GmNARK-mediated nodulation suppression. Plant Cell Physiol. 2011;52(9):1613–27.
Song XF, Guo P, Ren SC, Xu TT, Liu CM. Antagonistic peptide technology for functional dissection of CLV3/ESR genes in Arabidopsis. Plant Physiol. 2013;161(3):1076–85.
Stahl Y, Grabowski S, Bleckmann A, Kuhnemuth R, Weidtkamp-Peters S, Pinto KG, et al. Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr Biol. 2013;23(5):362–71.
Araya T, Miyamoto M, Wibowo J, Suzuki A, Kojima S, Tsuchiya YN, et al. CLE-CLAVATA peptide-receptor signaling module regulates the expansion of plant root system in a nitrogen-dependent manner. Proc Natl Acad Sci U S A. 2014;111(5):2029–34.
Fiume E, Fletcher JC. Regulation of Arabidopsis embryo and endosperm development by the polypeptide signaling molecule CLE8. Plant Cell. 2012;24(3):1000–12.
Wang X, Mitchum MG, Gao B, Li C, Diab H, Baum TJ, et al. A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana. Mol Plant Pathol. 2005;6:187–91.
Replogle A, Wang J, Bleckmann A, Hussey RS, Baum TJ, Sawa S, et al. Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE. Plant J. 2010;65:430–40.
Replogle A, Wang J, Paolillo V, Smeda J, Kinoshita A, Durbak A, et al. Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis. Mol Plant Microbe Interact. 2013;26:87–96.
Whitford R, Fernandez A, De Grood R, Ortega E, Hilson P. Plant CLE peptides from two distinct functional classes synergistically induce division of vascular cells. Proc Natl Acad Sci U S A. 2008;105(47):18625–30.
Hirakawa Y, Kondo Y, Fukuda H. TDIF peptide signaling regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis. Plant Cell. 2010;22(8):2618–29.
Etchells JP, Mishra LS, Kumar M, Campbell L, Turner SR. Wood formation in tree is increased by manipulating PXY-regulated cell division. Curr Biol. 2015;25(8):1050–5.
Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science. 2006;313(5793):1596–604.
Meng L, Ruth KC, Fletcher JC, Feldman L. The role of different CLE domains in Arabidopsis CLE polypeptide activity and functional specificity. Mol Plant. 2010;3(4):760–72.
Gao X, Guo Y. CLE peptides in plants: Proteolytic processing, structure-activity relationship, and ligand-receptor interaction. J Integr Plant Biol. 2012;54(10):738–45.
Matsubayashi Y. Small post-translationally modified peptide signals in Arabidopsis. The Arabidopsis Book. 2011;9:e0150. 10.1199:tab.0150.
Fiers M, Golemiec E, van der Schors R, van der Geest L, Li KW, Stiekema WJ, et al. The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences. Plant Physiol. 2006;141:1284–92.
Song XF, Yu DL, Xu TT, Ren SC, Guo P, Liu CM. Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis. Mol Plant. 2012;5(2):15–23.
Kondo T, Sawa S, Kinoshita A, Mizuno S, Kakimoto T, Fukuda H, et al. A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis. Science. 2006;313:845–8.
Ohyama K, Shinohara H, Ogawa-Ohnishi M, Matsubayashi Y. A glycopeptide regulating stem cell fate in Arabidopsis thaliana. Nat Chem Biol. 2009;5(8):578–80.
Xu TT, Song XF, Ren SC, Liu CM. The sequence flanking the N-terminus of CLV3 peptide is critical for its cleavage and activity in stem cell regulation in Arabidopsis. BMC Plant Biol. 2013;13:225.
Ni J, Clark SE. Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain. Plant Physiol. 2006;140(2):726–33.
Sawa S, Kinoshita A, Nakanomyo I, Fukuda H. CLV3/ESR-related (CLE) peptides as intercellular signaling molecules in plants. Chem Rec. 2006;6(6):303–10.
Hirakawa Y, Kondo Y, Fukuda H. Establishment and maintenance of vascular cell communities through local signaling. Curr Opin Plant Biol. 2011;14:17–23.
Sharma VK, Ramirez J, Fletcher JC. The Arabidopsis CLV3-like (CLE) genes are expressed in diverse tissues and encode secreted proteins. Plant Mol Biol. 2003;51(3):415–25.
Strabala TJ, O’donnell PJ, Smit AM, Ampomah-Dwamena C, Martin EJ, Netzler N, et al. Gain-of function phenotypes of many CLAVATA3/ESR genes, including four new family members correlated with tandem variations in the conserved CLAVATA3/ESR domain. Plant Physiol. 2006;140(4):1331–44.
Kinoshita A, Nakamura Y, Sasaki E, Kyozuka J, Fukuda H, Sawa S. Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-Related (CLE) peptides in Arabidopsis thaliana and Oryza sativa. Plant Cell Physiol. 2007;48(12):1821–5.
Jun J, Fiume E, Roeder AH, Meng L, Sharma VK, Osmont KS, et al. Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis. Plant Physiol. 2010;154(4):1721–36.
Kondo Y, Hirakawa Y, Kieber JJ, Fukuda H. CLE peptides can negatively regulate protoxylem vessel formation via cytokinin signaling. Plant Cell Physiol. 2011;52(1):37–48.
Bleckmann A, Weidtkamp-Peters S, Seidel CA, Simon R. Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane. Plant Physiol. 2010;152(1):166–76.
Guo Y, Han L, Hymes M, Denver R, Clark SE. CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification. Plant J. 2010;63(6):889–900.
Kinoshita A, Betsuyaku S, Osakabe Y, Mizuno S, Nagawa S, Stahl Y, et al. RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis. Development. 2010;137(22):3911–20.
Zhu Y, Wang Y, Li R, Song X, Wang Q, Huang S, et al. Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis. Plant J. 2010;61(2):223–33.
Petre B, Hacquard S, Duplessis S, Rouhier N. Genome analysis of poplar LRR-RLP gene clusters reveals RISP, a defense-related gene coding a candidate endogenous peptide elicitor. Front Plant Sci. 2014;5:111.
Zan Y, Ji Y, Zhang Y, Yang S, Song Y, Wang J. Genome-wide identification, characterization and expression analysis of Populus leucine-rich repeat receptor-like protein kinase genes. BMC Genomics. 2013;14:318.
Liu B, Wang L, Zhang J, Liu J, Zheng H, Chen J, et al. WUSCHEL-related Homeobox genes in Populus tomentosa: diversified expression patterns and a functional similarity in adventitious root formation. BMC Genomics. 2014;15:296.
Růžička K, Ursache R, Hejátko J, Helariutta Y. Xylem development-from the cradle to the grave. New Phytol. 2015;207(3):519–35.
Toufighi K, Brady SM, Austin R, Ly E, Provart NJ. The botany array resource: e-Northerns, expression angling, and promoter analyses. Plant J. 2005;43:153–63.
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012;40(Database issue):D1178–86.
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–8.
Crooks GE, Hon G, Chandonia JM, Brenner SE. Weblogo: A sequence logo generator. Genome Res. 2004;14(6):1188–90.
Guo AY, Zhu QH, Chen X, Luo JC. GSDS: a gene structure display server. Yi Chuan. 2007;29(8):1023–6.
Sjodin A, Street NR, Sandberg G, Gustafsson P, Jansson S. The Populus genome integrative explorer (PopGenIE): a new resource for exploring the Populus genome. New Phytol. 2009;182(4):1013–25.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25(24):4876–82.
Hall BG. Building phylogenetic tree from molecular data with MEGA. Mol Biol Eol. 2013;30(5):1229–35.
Gautier L, Cope L, Bolstad BM, Irizarry RA. Affy-analysis of Affymetrix Gene Chip data at the probe level. Bioinformatics. 2004;20(3):307–15.
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003;4(2):249–64.
Diboun I, Wernisch L, Orengo CA, Koltzenburg M. Microarray analysis after RNA amplification can detect pronounced differences in gene expression using limma. BMC Genomics. 2006;7:252.
Bender R, Lange S. Adjusting for multiple testing—when and how? J Clin Epidemiol. 2001;54(4):343–9.
We thank Dr. Guo and Dr. Griffith for critical reading of the manuscript, Dr. Wu and Dr. He for helpful discussions. Research in our group was supported by the National Natural Science Foundation of China (31271575; 31200902), by the Fundamental Research Funds for the Central Universities (GK201103005), by the Specialized Research Fund for the Doctoral Program of Higher Education from the Ministry of Education of China (20120202120009) and by the Natural Science Basic Research Plan in Shaanxi Province of China (2014JM3064).
The authors declare that they have no competing interests.
HH performed the data mining, data analysis and participated in the drafting of the manuscript. GZ and MW assisted to the data analysis. GW conceived the study, coordinated the research and wrote the manuscript. All authors read and approved the final manuscript.
A list of full-length sequences of all PtCLE proteins. The signal peptide cleavage sites of every PtCLEs are indicated. (PDF 27 kb)
The multiple alignment of all full-length PtCLE proteins. The C-terminal CLE motifs of each PtCLE were boxed. (PDF 38 kb)
The multiple sequence alignment of the CLE motifs derived from PtCLE proteins. The conserved residues are shaded in grey. (PDF 28 kb)
The multiple sequence alignment of all AtCLE and PtCLE proteins using their full-length proteins. The CLE motifs were boxed in red. The conserved residues are shaded in grey. (PDF 46 kb)
The multiple sequence alignment of all AtCLE and PtCLE proteins using their CLE motifs. The conserved residues are shaded in grey. (PDF 14 kb)
The multiple sequence alignment of all PtCLE proteins using their CLE motifs and five N-terminal residues flanking the CLE motifs (18-AA in length). The conserved residues are shaded in grey. Weblogo plot was used for graphical representation of the multiple sequence alignment of the 18-AA fragments. (PDF 72 kb)
The multiple sequence alignment of all AtCLE and PtCLE proteins using their CLE motifs and five N-terminal residues flanking the CLE motifs (18-AA in length). The conserved residues are shaded in grey. Weblogo plot was used for graphical representation of the multiple sequence alignment of the 18-AA fragments. (PDF 58 kb)
Phylogenetic analysis of PtCLE proteins by the Neighbor-joining method with 1000 bootstrap iterations. The tree was constructed using full-length PtCLE proteins. The percentage of trees in which the associated clades clustered together is shown (>40 %). (PDF 107 kb)
A list of AtCLE and PtCLE proteins with identical CLE motifs. (PDF 12 kb)
Phylogenetic analysis of AtCLE and PtCLE proteins by the Neighbor-joining method with 1000 bootstrap iterations. The tree was constructed using the conserved CLE motifs. The percentage of trees in which the associated clades clustered together is shown (>40 %). (PDF 70 kb)
Phylogenetic analysis of AtCLE and PtCLE proteins by the Neighbor-joining method with 1000 bootstrap iterations. The tree was constructed using full-length proteins. The percentage of trees in which the associated clades clustered together is shown (>40 %). (PDF 39 kb)
Transctiptional profiling of PtCLE genes in various organs and tissues using the available microarray data. The microarray data were downloaded from GEO and normalized for analysis. Color scale represents log2 expression values. (PDF 2632 kb)
A list of microarray datasets used in this study. Note that available microarray data were derived from different Populus species other than P. trichocarpa. (PDF 31 kb)
The expression pattern of AtCLE genes in shoot- and vascular-related tissues. Gene expression is displayed as normalized log2-transformed values. (PDF 148 kb)
The EST sequences and RNA-seq data for PtCLE genes which are not presented in the microarray. The expression level for RNA-seq data was presented as numbers of fragments per kilobase of exon in a gene per million fragments mapped (FPKM). (PDF 237 kb)
About this article
Cite this article
Han, H., Zhang, G., Wu, M. et al. Identification and characterization of the Populus trichocarpa CLE family. BMC Genomics 17, 174 (2016). https://doi.org/10.1186/s12864-016-2504-x