The CLE gene family in Populus trichocarpa

ABSTRACT

The CLE (CLAVATA3/Embryo Surrounding Region-related) peptides are small secreted signaling peptides that are primarily involved in the regulation of stem cell homeostasis in different plant meristems. Particularly, the characterization of the CLE41-PXY/TDR signaling pathway has greatly advanced our understanding on the potential roles of CLE peptides in vascular development and wood formation. Nevertheless, our knowledge on this gene family in a tree species is limited. In a recent study, we reported on a systematically investigation of the CLE gene family in Populus trichocarpa. The potential roles of PtCLE genes were studied by comparative analysis and transcriptional profiling. Among fifty PtCLE members, many PtCLE proteins share identical CLE motifs or contain the same CLE motif as that of AtCLEs, while PtCLE genes exhibited either comparable or distinct expression patterns comparing to their Arabidopsis counterparts. These findings indicate the existence of both functional conservation and functional divergence between PtCLEs and their AtCLE orthologues. Our results provide valuable resources for future functional investigations of these critical signaling molecules in woody plants.

The CLE (CLAVATA3/Embryo Surrounding Region-related) peptide family is one of the best-studied secretory peptide families in plants. Each CLE protein contains a N-terminal signal peptide following by a variable domain, and a conserved C-terminal CLE motif.^1-3 It is reported that the CLE motif is the functional domain of CLE proteins, and which is proteolytically processed from the CLE precursor protein.^4-7 In addition, the post-translational modifications of the CLE motif are critical for the function of the CLE protein.⁶ The bioactive CLE peptide is thought to interact with membrane-bound LRR-RLKs/RLPs (Leucine-Rich Repeat Receptor-like Kinases/Proteins) in a non-cell-autonomous manner.^1,8,9

The CLE genes have been found in many plant species, whereas the roles of most identified CLE genes remained to be elucidated.^2,10-15 As the 2 most known members of CLE peptide family, AtCLV3 and AtCLE40 are key regulators in coordinating stem cell homeostasis in the SAM (Shoot Apical Meristem) and RAM (Root Apical Meristem), respectively.¹ Other than their functions in stem cell fate, it has been also revealed that the CLE peptides are important players in modulating various plant growth and developmental processes such as nodulation,^16-18 protoxylem formation,¹⁹ embryogenesis and endosperm development,²⁰ phloem formation,²¹ pollen growth,²² and lateral root expansion.²³ In addition, CLE genes have been found to perform various biological roles in response to environmental stimuli.²⁴

Accumulated evidence highlights an essential function of the CLE genes in the regulation of vascular development.²⁵ A number of AtCLE genes have been shown to play pivotal roles in vascular development.^5,19,21,26 Particularly, both exogenous peptide application and overexpression of AtCLE41/TDIF resulted in promoted cambial cell proliferation and restricted xylem differentiation in a PXY/TDR-dependent manner.^26,27 Furthermore, co-overexpressing of PttCLE41 and PttPXY specifically in their endogenous expression domains generated poplar trees with increased wood formation and biomass production, suggesting that the CLE41/TDIF-PXY/TDR signaling module is evolutionarily conserved between herbaceous and woody species.²⁸ This work implies that to manipulate genes that are involved in the regulation of vasculature could greatly promote wood formation and biomass yield. Additionally, these results highlight the potential roles of CLE genes in the regulation of secondary growth and wood formation.

Despite these advances on the studies of CLE genes, our knowledge on this family in a tree species is fragmented. To this end, we undertook a genome-wide analysis in Populus trichocarpa to identify CLE genes and gain additional insights into their potential roles in various aspects of poplar growth and development, focusing on CLE genes exhibiting expression in vascular tissues which might be important for wood formation.¹¹ A total of 50 PtCLE proteins were identified from Populus trichocarpa and classified into 4 major groups. In-depth sequence analysis enabled us to identify PtCLE homologous copies in poplar and PtCLE orthologous copies between poplar and Arabidopsis.¹¹ In order to determine PtCLE candidate(s) that may function in shoot organogenesis, secondary growth and wood formation, the potential roles of PtCLE genes were studied by comparative analysis and transcriptional profiling. Transcriptomic analysis revealed that PtCLE genes generally were differentially expressed while some PtCLE genes exhibited tissue-specific expression patterns.¹¹ The presence of multiple PtCLEs in a single tissue type, exhibiting overlapping expression patterns, implies that the widespread redundancies among PtCLE genes. In comparison to their Arabidopsis counterparts, PtCLE genes exhibited either similar or different expression patterns, implying functional conservation in some cases and functional divergence in others.¹¹ Intriguingly, a group of 4 PtCLE proteins (PtCLE3/PtCLE12/PtCLE14/PtCLE38) shares an identical CLE motif as that of AtCLE41/TDIF. The expression patterns of PtCLE3 and PtCLE12 are similar to that of AtCLE41/TDIF. Altogether, these results imply that the existence of multiple CLE-RLK signaling pathways to modulate the secondary growth and wood formation in poplar.¹¹ In silico gene expression study also revealed that a number of PtCLE genes are predominantly expressed in various vascular tissues other than cambium, e.g. secondary phloem, developing xylem and mature xylem tissues,¹¹ suggesting diverse roles of PtCLE genes and a highly complicated regulatory network for the poplar vascular development. Collectively, our findings provide new insights on the sequence, structure, evolution, expression and function of PtCLE genes.

Previously we assessed potential roles of PtCLE proteins based on the phylogenetic relationships between AtCLEs and PtCLEs.¹¹ Our phylogenetic analysis indicated that some PtCLE proteins were closely related to their predicted Arabidopsis counterparts, which allowed interspecies identification of putative functional orthologs. Particularly, some PtCLE proteins contain identical CLE motifs as that of well-known AtCLE proteins, strongly supporting functional conservation between these CLE proteins and high-degree of functional redundancies. By using this approach, we extend our studies to identify PtCLEs sharing identical or nearly identical CLE motifs with 2 well-characterized AtCLEs, AtCLE40 and AtCLE45.^21,22,29

It has been shown that AtCLE40 can trigger the consumption of root meristem through the CLE40-ACR4-WOX5 pathway.²⁹ Recently, AtCLE40 was found to control the root meristem antagonistically with the CRN/CLV2 signaling pathway.³⁰ Although none of the PtCLE proteins comprise an identical CLE motif as that of AtCLE40, PtCLE1 shared a highly conserved CLE domain with AtCLE40 as only one residue is different (Fig. 1A). Nevertheless, PtCLE1 is preferentially expressed in vascular tissues,¹¹ suggesting that PtCLE1 may achieve a distinct role. Thus it is of interest to study the function of PtCLE1 by functional genomics tools and transgenic approaches.

Figure 1.

The CLE motif sequence alignment of PtCLE proteins with AtCLE40 (A) and AtCLE45 (B), respectively. The tree was constructed using the conserved CLE motifs by the Neighbor-joining method with 1000 bootstrap iterations. The identical residues are shaded in black while similar residues are shaded in gray.

Exogenous application of the AtCLE45 peptide suppressed Arabidopsis protophloem differentiation and root growth, which is inhibited in the bam3 mutant, suggesting that BAM3 mediated the suppression of protophloem differentiation and root meristem growth through CLE45.²¹ In addition, AtCLE45 alleviated heat stress via binding with SKM1/SKM2 to sustain pollen growth under higher temperatures, leading to successful seed production.²² PtCLE33 harbors an identical CLE motif to that of AtCLE45 (Fig. 1B), which advocates for the idea that PtCLE33, depending on its expression domain, may have a similar function as what has been shown for AtCLE45.

Recently, Liu et al. (2016) also reported the identification of 50 CLE genes in Populus trichocarpa, which is consistence with our results.^11,13 The roles of several PtCLE genes, that are potential counterparts of the well-studied AtCLV3 and AtCLE41/TDIF genes, were investigated by gain-of-function analysis and exogenous application of chemically synthesized CLE peptides.¹³ Over-expression of PtCLE genes and in vitro PtCLE peptide application assay in Arabidopsis resulted in nearly identical phenotypes,¹³ which are similar to what have been described for many AtCLE genes in Arabidopsis.^7,26,31-33 It has been reported that, regardless of their expression domains, the CLE motifs of CLE proteins largely determine the functional specificities of CLE proteins.^7,31-33 The fact that manipulation of multiple PtCLE genes or exogenously applied PtCLE peptides resulted in nearly identical phenotypes, argues the biological relevance of the obtained results and makes studies on the functional specificities of PtCLE genes even more challenging. Therefore, the obtained phenotypes are predictable, since overexpression of CLE genes or exogenous application of CLE peptides often causes pleiotropic phenotypes which possibly are the results of non-specific responses to exogenously abundant peptides acting through multiple signaling pathways. For instance, overexpression of many AtCLE genes leads to SAM termination.³³ However, there is no support that any ligand other than AtCLV3 is perceived by the receptor complexes.^8,34 Therefore, it remains unclear whether the observed phenotypes in Liu et al. (2016) are biologically relevant.

Fifty PtCLE genes have been identified in Populus trichocarpa genome.^11,13 A deep understanding of the roles of these PtCLE genes is thus essential in the future. The bottlenecks in determining the function of PtCLE genes are the lack of loss-of-function mutants and the high-level of functional redundancies. To cope with these difficulties, antagonistic peptide technology, RNAi approach and CRISPR/Cas9 technology are necessary to apply in practice.^22,35 In addition to the conserved CLE motif, the specificity of CLE gene is depend on its expression pattern and level.^7,34,36 To gain an in-depth understanding of how functional specificity of the PtCLE is determined, the promoter-driven reporter lines of each PtCLE gene, similar to that have been done for AtCLE genes,³⁴ are essentially generated to obtain high-resolution expression data. Our transcriptional analysis indicates that PtCLE genes may involve in many aspects of poplar growth and development other than meristem maintenance,¹¹ in which they are likely recognized by different receptors. As CLE ligands are normally perceived by LRR-RLKs/RLPs to modulate diverse biological processes,^3,8,9 it would be intriguing to identify their cognate receptor(s) for the interested PtCLE peptide. Taken together, our study provides essential insights for better understanding of the roles of CLE genes in a tree species through comparative analysis and transcriptional profiling. The evolutionarily conserved CLE peptides between Arabidopsis and poplar might help to dissect the roles of PtCLE genes. Nevertheless, the future challenge still will be functional dissection of PtCLE genes and integrating their functions into the complex regulatory networks.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Dr. Long (Laboratoire de Reproduction et Développement des Plantes, CNRS, INRA, ENS Lyon, UCBL, Université de Lyon, France) for critical reading of the article. Work in our group is supported by the National Natural Science Foundation of China (31271575; 31200902), the Fundamental Research Funds for the Central Universities (GK201103005), the Specialized Research Fund for the Doctoral Program of Higher Education from the Ministry of Education of China (20120202120009), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Natural Science Basic Research Plan in Shaanxi Province of China (2014JM3064).