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1.  CLE genes may act in a variety of tissues/cells and involve other signaling cascades in addition to CLV3-WUS-like pathways 
Plant Signaling & Behavior  2011;6(1):105-108.
CLE, which is the term for the CLV3/ESR-related gene family, is thought to participate in CLAVATA3-WUSCHEL (CLV3-WUS) and CLV3-WUS-like signaling pathways to regulate meristem activity in plant. Although some CLE genes are expressed in meristems, many CLE genes appear to express in a variety of tissues/cells. Here we report that CLE14 and CLE20 express in various specific tissues/cells outside the shoot/root apical meristem (SAM/RAM), including in highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles and involve other signaling cascades in addition to the CLV3-WUS and CLV3-WUS-like pathways.
PMCID: PMC3122018  PMID: 21270538
CLE; CLAVATA3-WUSCHEL; cell signaling and development; root apical meristem; arabidopsis
2.  CLE14/CLE20 peptides may interact with CLAVATA2/CORYNE receptor-like kinases to irreversibly inhibit cell division in the root meristem of Arabidopsis 
Planta  2010;232(5):1061-1074.
Towards an understanding of the interacting nature of the CLAVATA (CLV) complex, we predicted the 3D structures of CLV3/ESR-related (CLE) peptides and the ectodomain of their potential receptor proteins/kinases, and docking models of these molecules. The results show that the ectodomain of CLV1 can form homodimers and that the 12-/13-amino-acid CLV3 peptide fits into the binding clefts of the CLV1 dimers. Our results also demonstrate that the receptor domain of CORYNE (CRN), a recently identified receptor-like kinase, binds tightly to the ectodomain of CLV2, and this likely leads to an increased possibility for docking with CLV1. Furthermore, our docking models reveal that two CRN-CLV2 ectodomain heterodimers are able to form a tetramer receptor complex. Peptides of CLV3, CLE14, CLE19, and CLE20 are also able to bind a potential CLV2-CRN heterodimer or heterotetramer complex. Using a cell-division reporter line, we found that synthetic 12-amino-acid CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the root apical meristem, resulting in a short-root phenotype. Intriguingly, we observed that exogenous application of cytokinin can partially rescue the short-root phenotype induced by over-expression of either CLE14 or CLE20 in planta. However, cytokinin treatment does not rescue the short-root phenotype caused by exogenous application of the synthetic CLE14/CLE20 peptides, suggesting a requirement for a condition provided only in living plants. These results therefore imply that the CLE14/CLE20 peptides may act through the CLV2-CRN receptor kinase, and that their availabilities and/or abundances may be affected by cytokinin activity in planta.
PMCID: PMC2940047  PMID: 20697738
CLE; 3D structures; Docking models; Root apical meristem; Cytokinin
3.  CLE Peptides can Negatively Regulate Protoxylem Vessel Formation via Cytokinin Signaling 
Plant and Cell Physiology  2010;52(1):37-48.
Cell–cell communication is critical for tissue and organ development. In plants, secretory CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides function as intercellular signaling molecules in various aspects of tissue development including vascular development. However, little is known about intracellular signaling pathways functioning in vascular development downstream of the CLE ligands. We show that CLE peptides including CLE10, which is preferentially expressed in the root vascular system, inhibit protoxylem vessel formation in Arabidopsis roots. GeneChip analysis displayed that CLE10 peptides repressed specifically the expression of two type-A Arabidopsis Response Regulators (ARRs), ARR5 and ARR6, whose products act as negative regulators of cytokinin signaling. The arr5 arr6 roots exhibited defective protoxylem vessel formation. These results indicate that CLE10 inhibits protoxylem vessel formation by suppressing the expression of type-A ARR genes including ARR5 and ARR6. This was supported by the finding that CLE10 did not suppress protoxylem vessel formation in a background of arr10 arr12, a double mutant of type-B ARR genes. Thus, our results revealed cross-talk between CLE signaling and cytokinin signaling in protoxylem vessel formation in roots. Taken together with the indication that cytokinin signaling functions downstream of the CLV3/WUS signaling pathway in the shoot apical meristem, the cross-talk between CLE and cytokinin signaling pathways may be a common feature in plant development.
PMCID: PMC3023848  PMID: 20802224
CLV3/ESR-related (CLE); cytokinin; signal transduction; protoxylem; Arabidopsis thaliana
4.  CLE peptide signaling during plant development 
Protoplasma  2009;240(1-4):33-43.
Peptide signaling in plants is a rapid developing area of research which focuses on so called peptide hormones. These signaling molecules are utilized for inter-cellular communication in different developmental processes, beside the usage of the more well-known phytohormones. Probably the best studied peptide ligands in plants are the CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins. This family of signaling polypeptides is comprised of 32 members in Arabidopsis and, with the exception of the presence of related proteins in some parasitic worms, is restricted to the plant kingdom. CLV3 is one of the founding CLE genes and is involved in stem cell niche maintenance in apical meristems during plant development. While the CLV signaling pathway is well characterized with the identification of three receptors and a stem-cell-promoting transcription factor as target, the functioning of other family members is not or poorly understood. The recent discoveries of a new type of receptor involved in CLV signaling and a functional pathway for CLE40 in root development mark the rapid progress that is made in the area of CLE peptide signaling. This review gives an overview how CLE peptides are used as signaling molecules, and how they are involved in cell-to-cell communication in concert with different known and unknown receptors in a range of developmental processes during plant development.
PMCID: PMC2841256  PMID: 20016993
Meristem development; CLE peptide; Ligand; Clavata
5.  A large family of genes that share homology with CLE domain in Arabidopsis and rice 
Plant Signaling & Behavior  2008;3(5):337-339.
Thirty-one CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins are encoded in the Arabidopsis genome, and they are supposed to function as dodecapeptides with two hydroxyproline residues. Twenty-six synthetic CLE peptides, corresponding to the predicted products of the 31 CLE genes, were examined in Arabidopsis and rice. Nineteen CLE peptides induced root meristem consumption, resulting in the short root phenotype in Arabidopsis and rice, whereas no CLE peptides affected the shoot apical meristem in rice. Database searches revealed 47 putative CLE genes in the rice genome. Three of the rice CLE genes, OsCLE502, OsCLE504 and OsCLE506, encode CLE proteins with multiple CLE domains, which are not found in the Arabidopsis genome, and polyproline region was found between these CLE domains. These results indicate conserved and/or diverse CLE functions in each plant species.
PMCID: PMC2634276  PMID: 19841664
CLE; CLAVATA; meristem; SAM; RAM; peptide
6.  CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification 
CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor-kinase CLV1 binds to the CLV3-derived CLE ligand. The biochemical role of receptor-like protein CLV2 has remained largely unknown. While genetic analysis suggested that CLV2, together with the membrane kinase CRN, act in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here we report evidence for distinct CLV2/CRN heteromultimeric and CLV1/BAM multimeric complexes in transient expression and in Arabidopsis. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane-localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3-derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 over-expression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand-binding receptor complexes controlling stem cell specification in Arabidopsis.
PMCID: PMC2974754  PMID: 20626648
CLAVATA2; CLE peptide; CORYNE; CLAVATA1; receptor complex; meristem
7.  Genome-wide characterization, expression and functional analysis of CLV3/ESR gene family in tomato 
BMC Genomics  2014;15(1):827.
By encoding a group of small secretory peptides, the members of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) family play important roles in cell-to-cell communication to control the balance between stem cell proliferation and differentiation in plant development. Despite recent identification and characterization of members of this gene family in several plant species, little is known about its functional role in plants with fleshy fruits.
In total, fifteen CLE genes (SlCLE1-15) were identified from tomato (Solanum lycopersicum cv. ‘Heinz-1706’) genome and their multiple characters including phylogeny, gene structures, chromosome locations, conserved motifs and cis-elements in the promoter sequences, were analyzed. Real-time PCR analysis showed that 13 out of 15 identified SlCLE genes are transcribed and exhibit remarkably unique expression patterns among tissues and organs. In particular, SlCLE12, the homologue of Arabidopsis CLE41/44 gene, appears to be the dominant CLE gene in most of tested tissues with its maximum expression found in vascular tissues. Meanwhile, SlCLE1, 10, 13 exhibit specific but distinct expression in flower bud, root and shoot apex, respectively. More notably, several SlCLEs are dramatically regulated in their transcriptional levels during fruit development and ripening, indicating significant role these genes may potentially play in the critical physiological process. Upon the treatment with synthetic peptides corresponding to the 12-aa CLE domains of SlCLE 10, 12 and 13, tomato seedlings exhibit a clear reduction in root length to varying degrees.
This study provides a comprehensive genomic analysis of CLE gene family in tomato, a crop species with fleshy fruit. Differential expression patterns of various SlCLEs provide important insights into the functional divergence of CLE signaling cascade in Solanaceae species, especially their potential involvements in the regulation of fruit development and ripening.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-827) contains supplementary material, which is available to authorized users.
PMCID: PMC4195864  PMID: 25266499
Solanum lycopersicum; CLE gene family; Phylogenetic analysis; Tissue expression pattern; Peptide application
8.  Bioinformatic analysis of the CLE signaling peptide family 
BMC Plant Biology  2008;8:1.
Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases.
We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups.
Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity.
PMCID: PMC2254619  PMID: 18171480
9.  WUSCHEL-related Homeobox genes in Populus tomentosa: diversified expression patterns and a functional similarity in adventitious root formation 
BMC Genomics  2014;15:296.
WUSCHEL (WUS)-related homeobox (WOX) protein family members play important roles in the maintenance and proliferation of the stem cell niche in the shoot apical meristem (SAM), root apical meristem (RAM), and cambium (CAM). Although the roles of some WOXs in meristematic cell regulation have been well studied in annual plants such as Arabidopsis and rice, the expression and function of WOX members in woody plant poplars has not been systematically investigated. Here, we present the identification and comprehensive analysis of the expression and function of WOXs in Populus tomentosa.
A genome-wide survey identified 18 WOX encoding sequences in the sequenced genome of Populus trichocarpa (PtrWOXs). Phylogenetic and gene structure analysis revealed that these 18 PtrWOXs fall into modern/WUS, intermediate, and ancient clades, but that the WOX genes in P. trichocarpa may have expanded differently from the WOX genes in Arabidopsis. In the P. trichocarpa genome, no WOX members could be closely classified as AtWOX3, AtWOX6, AtWOX7, AtWOX10, and AtWOX14, but there were two copies of WOX genes that could be classified as PtrWUS, PtrWOX2, PtrWOX4, PtrWOX5, PtrWOX8/9, and PtrWOX11/12, and three copies of WOX genes that could be classified as PtrWOX1 and PtrWOX13. The use of primers specific for each PtrWOX gene allowed the identification and cloning of 18 WOX genes from P. tomentosa (PtoWOXs), a poplar species physiologically close to P. trichocarpa. It was found that PtoWOXs and PtrWOXs shared very high amino acid sequence identity, and that PtoWOXs could be classified identically to PtrWOXs. We revealed that the expression patterns of some PtoWOXs were different to their Arabidopsis counterparts. When PtoWOX5a and PtoWOX11/12a, as well as PtoWUSa and PtoWOX4a were ectopically expressed in transgenic hybrid poplars, the regeneration of adventitious root (AR) was promoted, indicating a functional similarity of these four WOXs in AR regeneration.
This is the first attempt towards a systematical analysis of the function of WOXs in P. tomentosa. A diversified expression, yet functional similarity of PtoWOXs in AR regeneration is revealed. Our findings provide useful information for further elucidation of the functions and mechanisms of WOXs in the development of poplars.
PMCID: PMC4023605  PMID: 24750781
Adventitious root; Expression; Homeobox; Populus; WOX; Wuschel-related
10.  Is the Arabidopsis root niche protected by sequestration of the CLE40 signal by its putative receptor ACR4? 
Plant Signaling & Behavior  2009;4(7):634-635.
A tight but also dynamic regulation is necessary to control the size of stem cell populations in response to internal and external cues. The stem cells of the Arabidopsis shoot and root meristems are governed by the niche cells of the organizing centre (OC) and the quiescent centre (QC), respectively. The well characterized CLV3/WUS negative feedback loop adjusts homeostasis of the stem cell population in the shoot. Here, the CLAVATA3 (CLV3) dodecapeptide, expressed by the stem cells, signals to repress WUSCHEL (WUS), which is expressed in the subjacent OC cells, and in turn activates CLV3 expression non-cell autonomously. However, a similar signaling module controlling the root stem cell population was as yet unknown. In the June issue of Current Biology we report on such a signaling module comprising CLE40 (a CLV3 homologue) that acts via the receptor kinase Arabidopsis Crinkly4 (ACR4) to repress the WUS homologue WOX5 which maintains distal root stem cells. Furthermore, we showed that CLE40 peptide (CLE40p) treatment upregulates ACR4 expression. In this Addendum, we are further elaborating our hypothesis in which the upregulation of ACR4 as a consequence of ectopic CLE40p builds a protective barrier for the QC niche cells.
PMCID: PMC2710560  PMID: 19820344
arabidopsis; root meristem; niche; stem cells; QC; CLE40; ACR4; WOX5
11.  Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and the CLE-LIKE signal peptide genes in the Pinophyta 
BMC Plant Biology  2014;14:47.
There is a rapidly growing awareness that plant peptide signalling molecules are numerous and varied and they are known to play fundamental roles in angiosperm plant growth and development. Two closely related peptide signalling molecule families are the CLAVATA3-EMBRYO-SURROUNDING REGION (CLE) and CLE-LIKE (CLEL) genes, which encode precursors of secreted peptide ligands that have roles in meristem maintenance and root gravitropism. Progress in peptide signalling molecule research in gymnosperms has lagged behind that of angiosperms. We therefore sought to identify CLE and CLEL genes in gymnosperms and conduct a comparative analysis of these gene families with angiosperms.
We undertook a meta-analysis of the GenBank/EMBL/DDBJ gymnosperm EST database and the Picea abies and P. glauca genomes and identified 93 putative CLE genes and 11 CLEL genes among eight Pinophyta species, in the genera Cryptomeria, Pinus and Picea. The predicted conifer CLE and CLEL protein sequences had close phylogenetic relationships with their homologues in Arabidopsis. Notably, perfect conservation of the active CLE dodecapeptide in presumed orthologues of the Arabidopsis CLE41/44-TRACHEARY ELEMENT DIFFERENTIATION (TDIF) protein, an inhibitor of tracheary element (xylem) differentiation, was seen in all eight conifer species. We cloned the Pinus radiata CLE41/44-TDIF orthologues. These genes were preferentially expressed in phloem in planta as expected, but unexpectedly, also in differentiating tracheary element (TE) cultures. Surprisingly, transcript abundances of these TE differentiation-inhibitors sharply increased during early TE differentiation, suggesting that some cells differentiate into phloem cells in addition to TEs in these cultures. Applied CLE13 and CLE41/44 peptides inhibited root elongation in Pinus radiata seedlings. We show evidence that two CLEL genes are alternatively spliced via 3′-terminal acceptor exons encoding separate CLEL peptides.
The CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species. Only one CLE peptide sequence has been 100% conserved between gymnosperms and angiosperms over 300 million years of evolutionary history, the CLE41/44-TDIF peptide and its likely conifer orthologues. The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants. Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.
PMCID: PMC4016512  PMID: 24529101
CLE peptide ligands; CLEL peptide ligands; Pinophyta; Conifers; Phylogenetic analysis; Pine tracheary element system
12.  Plant Vascular Cell Division Is Maintained by an Interaction between PXY and Ethylene Signalling 
PLoS Genetics  2012;8(11):e1002997.
The procambium and cambium are meristematic tissues from which vascular tissue is derived. Vascular initials differentiate into phloem towards the outside of the stem and xylem towards the inside. A small peptide derived from CLV-3/ESR1-LIKE 41 (CLE41) is thought to promote cell divisions in vascular meristems by signalling through the PHLOEM INTERCALLATED WITH XYLEM (PXY) receptor kinase. pxy mutants, however, display only small reductions in vascular cell number, suggesting a mechanism exists that allows plants to compensate for the absence of PXY. Consistent with this idea, we identify a large number of genes specifically upregulated in pxy mutants, including several AP2/ERF transcription factors. These transcription factors are required for normal cell division in the cambium and procambium. These same transcription factors are also upregulated by ethylene and in ethylene-overproducing eto1 mutants. eto1 mutants also exhibit an increase in vascular cell division that is dependent upon the function of at least 2 of these ERF genes. Furthermore, blocking ethylene signalling using a variety of ethylene insensitive mutants such as ein2 enhances the cell division defect of pxy. Our results suggest that these factors define a novel pathway that acts in parallel to PXY/CLE41 to regulate cell division in developing vascular tissue. We propose a model whereby vascular cell division is regulated both by PXY signalling and ethylene/ERF signalling. Under normal circumstances, however, PXY signalling acts to repress the ethylene/ERF pathway.
Author Summary
Plants transport water and nutrients throughout their bodies using a specialised vascular system. Vascular tissue is also responsible for providing structural support to plants; for example, wood is made up of specialised vascular cells. Consequently, the vascular system constitutes the majority of plant biomass. Chemicals from plant biomass could be used to make the next generation of biofuels in order to reduce dependence on fossil fuels. Vascular tissue is derived from a group of dividing cells present in a structure called the procambium, but mechanisms controlling cell division in this structure remain poorly understood. Understanding the events that occur in the procambium may help us to understand how we can best utilise plants for increased plant biomass, for example, for biofuel and wood production. We have identified a number of genes that regulate cell division in the procambium that are controlled by the gaseous plant hormone ethylene. We show that ethylene signalling, in turn, interacts with PXY, a gene encoding a signalling component that also controls vascular cell division. Our results demonstrate that the interaction between ethylene and PXY signalling is responsible for maintaining the plant vascular system.
PMCID: PMC3499249  PMID: 23166504
13.  FON2 SPARE1 Redundantly Regulates Floral Meristem Maintenance with FLORAL ORGAN NUMBER2 in Rice 
PLoS Genetics  2009;5(10):e1000693.
CLAVATA signaling restricts stem cell identity in the shoot apical meristem (SAM) in Arabidopsis thaliana. In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2), closely related to CLV3, is involved as a signaling molecule in a similar pathway to negatively regulate stem cell proliferation in the floral meristem (FM). Here we show that the FON2 SPARE1 (FOS1) gene encoding a CLE protein functions along with FON2 in maintenance of the FM. In addition, FOS1 appears to be involved in maintenance of the SAM in the vegetative phase, because constitutive expression of FOS1 caused termination of the vegetative SAM. Genetic analysis revealed that FOS1 does not need FON1, the putative receptor of FON2, for its action, suggesting that FOS1 and FON2 may function in meristem maintenance as signaling molecules in independent pathways. Initially, we identified FOS1 as a suppressor that originates from O. sativa indica and suppresses the fon2 mutation in O. sativa japonica. FOS1 function in japonica appears to be compromised by a functional nucleotide polymorphism (FNP) at the putative processing site of the signal peptide. Sequence comparison of FOS1 in about 150 domesticated rice and wild rice species indicates that this FNP is present only in japonica, suggesting that redundant regulation by FOS1 and FON2 is commonplace in species in the Oryza genus. Distribution of the FNP also suggests that this mutation may have occurred during the divergence of japonica from its wild ancestor. Stem cell maintenance may be regulated by at least three negative pathways in rice, and each pathway may contribute differently to this regulation depending on the type of the meristem. This situation contrasts with that in Arabidopsis, where CLV signaling is the major single pathway in all meristems.
Author Summary
The body plan of plants is regulated by the function of apical meristems that are generated in the embryo. Leaves and floral organs are derived from cells supplied by stem cells in the vegetative shoot apical meristem (SAM) and the floral meristem (FM), respectively. Thus, genetic regulation of stem cell maintenance is a central issue in plant development. In the model plant Arabidopsis thaliana, CLAVATA3 (CLV3) functions as a key signaling molecule to restrict the size of the stem cell population in both the SAM and the FM. In rice, however, we show here that two CLV3-like genes, FLORAL ORGAN NUMBER2 (FON2) and FON2 SPARE1 (FOS1), redundantly regulate maintenance of the FM. We also show that FOS1 is likely to be involved in maintenance of the vegetative SAM, whereas FON2 plays no role in regulation in this meristem. FOS1 appears to act via a putative receptor that differs from the FON2 receptor, suggesting that these two signaling molecules function in independent pathways to restrict stem cells in different ways depending on the type of meristem. In addition, we show that the FOS1 gene was compromised in the standard rice, Oryza sativa spp. japonica, during the evolution of rice.
PMCID: PMC2752996  PMID: 19834537
14.  WOX4 and WOX14 act downstream of the PXY receptor kinase to regulate plant vascular proliferation independently of any role in vascular organisation 
Development (Cambridge, England)  2013;140(10):2224-2234.
In plants, the cambium and procambium are meristems from which vascular tissue is derived. In contrast to most plant cells, stem cells within these tissues are thin and extremely long. They are particularly unusual as they divide down their long axis in a highly ordered manner, parallel to the tangential axis of the stem. CLAVATA3-LIKE/ESR-RELATED 41 (CLE41) and PHLOEM INTERCALATED WITH XYLEM (PXY) are a multifunctional ligand-receptor pair that regulate vascular cell division, vascular organisation and xylem differentiation in vascular tissue. A transcription factor gene, WUSCHEL HOMEOBOX RELATED 4 (WOX4) has been shown to act downstream of PXY. Here we show that WOX4 acts redundantly with WOX14 in the regulation of vascular cell division, but that these genes have no function in regulating vascular organisation. Furthermore, we identify an interaction between PXY and the receptor kinase ERECTA (ER) that affects the organisation of the vascular tissue but not the rate of cell division, suggesting that cell division and vascular organisation are genetically separable. Our observations also support a model whereby tissue organisation and cell division are integrated via PXY and ER signalling, which together coordinate development of different cell types that are essential for normal stem formation.
PMCID: PMC3912870  PMID: 23578929
Arabidopsis; Cell division; Phloem; Procambium; Signalling; Vascular
15.  CLE polypeptide signaling gene expression in Arabidopsis embryos 
Plant Signaling & Behavior  2011;6(3):443-444.
The CLAVATA3 (CLV3)/ESR-related (CLE) family of small polypeptides mediate intercellular signaling events in plants. The biological roles of several CLE family members have been characterized, but the function of the majority still remains elusive. We recently performed a systematic expression analysis of 23 Arabidopsis CLE genes to gain insight into the developmental processes they may potentially regulate during vegetative and reproductive growth. Our study revealed that each Arabidopsis tissue expresses one or more CLE genes, suggesting that they might play roles in many developmental and/or physiological processes. Here we determined the expression patterns of nine Arabidopsis CLE gene promoters in mature embryos and compared them to the known expression patterns in seedlings. We found that more than half of these CLE genes have similar expression profiles at the embryo and seedling stages, whereas the rest differ dramatically. The implications of these findings in understanding the biological processes controlled by these CLE genes are discussed.
PMCID: PMC3142434  PMID: 21278487
arabidopsis; CLE; embryo; polypeptide; signaling
16.  Membrane distributions of two ligand-binding receptor complexes in the CLAVATA pathway 
Plant Signaling & Behavior  2010;5(11):1442-1445.
Genetic studies have suggested that transmembrane proteins CLAVATA1 (CLV1), CLV2, CORYNE (CRN), BAM1 and BAM2 all play a role in relaying the CLV3 signal and thus regulating stem cell homeostasis at the shoot meristem (SM). The extracellular domain of CLV1 was previously shown to bind the CLE peptide derived from CLV3, providing direct evidence that CLV3-CLV1 function as a ligand-receptor pair. How the other putative receptors function in the CLV pathway, however, remained unclear. We demonstrated in a recent Plant Journal article that the receptor-like protein CLV2 and the receptor-kinases BAM1 and BAM2 also bind to the CLV3 CLE peptide ligand with an affinity similar to that of CLV1. Critically, these ligand binding receptors form two distinct complexes in both transient expression in tobacco and in Arabidopsis meristem cells: a CLV2/CRN multimer and a CLV1/BAM multimer. Here we examine in detail the subcellular membrane partitioning for the receptor proteins in transient expression by two-phase partitioning and co-expression with known subcellular markers. All tested proteins measurably accumulate at the plasma membrane. While CLV1 primarily co-localizes with a plasma membrane marker, CLV2 shows greater co-localization with an endoplasmic reticulum (ER) marker.
PMCID: PMC3115250  PMID: 21051944
CLAVATA2; CLAVATA1; CORYNE; subcellular localization; plasma membrane; endoplasmic reticulum; receptor complex; meristem
17.  Evolution of CLE signaling 
Plant Signaling & Behavior  2009;4(6):477-481.
The shoot apical meristem is maintained by the intercellular factor, CLV3, a dodecapeptide in Arabidopsis. CLV3 belongs to the CLE family and putative CLE genes have been found in various plants, even in the moss Physcomitrella patens. Here, we report that a pteridophyte, Selaginella moelendorffii, also has 15 putative CLE genes in its genome. On the other hand, CLV1 is reported to function as a receptor for the CLV3 peptide, and other CLE peptides might be recognized by CLV1 homologues in various plants. Recent genetic studies of the crn and sol2 mutants of Arabidopsis have revealed that SOL2/CRN encodes a receptor-like kinase protein. SOL2/CRN functions together with CLV2 independently of CLV1 in the CLE signaling pathway. Phylogenetic analysis of CLV1, CLV2 and SOL2/CRN revealed that Arabidopsis, rice, Populus trichocarpa and Vitis vinifera have one copy of the SOL2/CRN and CLV2 homologues, and Selaginella moelendorffii and Physcomitrella patens have no homologues. In contrast, a number of CLV1 homologues were identified in the genomic databases of Arabidopsis, rice, Populus trichocarpa, Vitis vinifera, and even a pteridophyte, Selaginella moelendorffii, and a moss, Physcomitrella patens. These results indicate that CLV1 and its homologues play multiple roles in plant development and environmental responses, whereas SOL2/CRN and CLV2 have more specific roles in vascular plants.
PMCID: PMC2688290  PMID: 19816140
SOL2; CLE; CLV; CRN; meristem; receptor kinase
18.  Signaling in the Arabidopsis shoot meristem stem cell niche correlates with ligand-dependent trafficking of the CLV1 receptor kinase 
Current biology : CB  2011;21(5):345-352.
Cell numbers in above-ground meristem types of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex [1]. CLV3 made in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 as well as WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. High-level overexpression of CLV3 eliminates the stem cells and causes meristem termination [2], and loss of CLV3 function allows meristem overproliferation [3]. There are many open questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype [4]. Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model which states that CLV3 is quantitatively bound by CLV1 in the apical regions of the meristem, allowing continued WUS activity in lower regions [5]. This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants.
PMCID: PMC3072602  PMID: 21333538
19.  Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean 
Journal of Experimental Botany  2011;62(13):4649-4659.
CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biological activity of Medicago truncatula CLE36, which possesses broadly conserved border sequences flanking the putative final active CLE36 peptide product, was assessed. Using in vitro root growth assays and an in vitro root and callus formation assay it is shown that CLE36 peptides of different lengths possess differential biological activities. Using mass spectrometry, Glycine max and Medicago extracellular fluids were each shown to possess an endoproteolytic activity that recognizes and cleaves at border sequences in a synthetic 31 amino acid CLE36 ‘propeptide bait’ to liberate biologically active peptide products. Inhibitor studies suggest that a subtilisin, in combination with a carboxypeptidase, liberated and trimmed CLE36, respectively, to form biologically relevant 11–15 amino acid cleavage products. The 15 amino acid cleavage product is more biologically potent on Arabidopsis than shorter or longer CLE peptides. In situ hybridization shows that the soybean orthologue of CLE36 (GmCLE34) is expressed in the provascular tissue. The results suggest that secreted subtilisins can specifically recognize the border sequences of CLE36 propeptides and liberate biologically active cleavage products. These secreted proteases may affect the stability and biological activity of CLE peptides in the apoplast or be involved in CLE36 processing.
PMCID: PMC3170558  PMID: 21633083
Arabidopsis; carboxypeptidase; CLE peptide; mass spectrometry; Medicago; proteomics; rice; secreted proteins; soybean; subtilisin
20.  The sequence flanking the N-terminus of the CLV3 peptide is critical for its cleavage and activity in stem cell regulation in Arabidopsis 
BMC Plant Biology  2013;13:225.
Although it is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown.
We developed a mass spectrometry (MS)-based in vitro assay to monitor the cleavage of heterologously produced CLV3 fusion protein. Through co-cultivation of the fusion protein with Arabidopsis seedlings, we identified two cleavage sites: the previously reported one before Arg70 and a new one before Met39. Using synthetic peptides together with MALDI-Tof-MS analyses, we demonstrated that the non-conserved 5-AA motifs flanking N-termini of the CLV3 and its orthologous CLE1 peptides were critical for their cleavages and optimal activities in vitro. We also found that substitutions of Leu69 by Ala in fusion protein and in synthetic peptide of CLV3 compromised their cleavages, leading to significantly reduced activities in regulating the sizes of shoot and root meristems.
These results suggest that 5-AA residues flanking the N-terminus of CLV3 peptide are required for proper cleavages and optimal function in stem cell regulation.
PMCID: PMC3878228  PMID: 24369789
CLV3; Peptide cleavage; Flanking sequence; AA substitution; Stem cell regulation
21.  Structure–function analysis of the GmRIC1 signal peptide and CLE domain required for nodulation control in soybean 
Journal of Experimental Botany  2013;64(6):1575-1585.
Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptide-encoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) of GmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation.
PMCID: PMC3617822  PMID: 23386683
Autoregulation of nodulation; CLE peptides; legumes; nodulation; soybean; symbiosis.
22.  Variable domain I of nematode CLEs directs post-translational targeting of CLE peptides to the extracellular space 
Plant Signaling & Behavior  2010;5(12):1633-1635.
Effector proteins expressed in the esophageal gland cells of cyst nematodes are delivered into plant cells through a hollow, protrusible stylet. Although evidence indicates that effector proteins function in the cytoplasm of the syncytium,1–3 technical constraints have made it difficult to directly determine where nematode effector proteins are initially delivered: cytoplasm, extracellular space, or both. Recently, we demonstrated that soybean cyst nematode CLE (HgCLE) propeptides are delivered to the cytoplasm of syncytial cells. Genetic and biochemical analyses indicate that the variable domain (VD) sequence is then required for targeting cytoplasmically delivered nematode CLEs to the apoplast where they function as ligand mimics of endogenous plant CLE peptides.4 The fact that nematode CLEs are targeted through the gland cell secretory pathway and delivered as mature propeptides into plant cells makes it impossible for these proteins to be subsequently delivered to the extracellular space via co-translational translocation through the endoplasmic reticulum (ER) secretory pathway of the host cell. However, when expressed in transgenic plants, if the mature nematode CLE propeptide harbored a functional cryptic signal peptide, it could possibly traffic to the apoplast through the ER secretory pathway by co-translational translocation. Here, we present evidence that VDI, the N-terminal sequence of the VD of HgCLE2,4 is sufficient for trafficking CLE peptides to the apoplast and that trafficking is indeed through an alternative pathway other than co-translational translocation.
PMCID: PMC3115119  PMID: 21150256
cyst nematode; effector; CLE; variable domain; trafficking; endoplasmic reticulum; co-translational translocation; post-translational
23.  DNA uptake by Arabidopsis induces changes in the expression of CLE peptides which control root morphology 
Plant Signaling & Behavior  2010;5(9):1112-1114.
The presence of externally supplied DNA in the growth medium enhances growth of lateral roots and root hairs in Arabidopsis. This phenomenon cannot be attributed to phosphorus (P) limitation because it is independent of the plants' P status. Rather, we hypothesized that DNA triggers a currently unknown signaling pathway. Analyzing the transcriptional changes of genes induced by externally supplied DNA, we show that 7 of the 17 studied CLAVATA3/ESR-related (CLEs) genes were differentially regulated. CLEs are known peptide hormones that affect plant development including root morphology. While previous research had shown that overexpression of these CLE genes alters root morphology, changes in gene expression had not been linked to environmental triggers. The differential expression of these CLEs genes and accompanied changes of the root phenotype are indicative of a DNA-elicited signal pathway which affects root development. We conclude that DNA acts as a signaling compound which induces root proliferation in a way that would enhance the plant's ability to acquire nutrients from soil organic matter. Our study further confirms the importance of CLEs for controlling root morphology in response to specific environmental conditions, and draws attention to a novel role of DNA as a signaling compound.
PMCID: PMC3115079  PMID: 21490419
arabidopsis; DNA; CLEs; root morphology; plant signaling
24.  The Shoot Apical Meristem Size Regulated by FON4 in Rice 
Plant Signaling & Behavior  2007;2(2):115-116.
CLAVATA pathway is one of best-characterized signaling pathway involves in the regulation of meristem development in Arabidopsis. Increasing evidence indicated that this pathway also exist in the monocots as well as in the dicots. We have recently identified FON4 in rice as an ortholog of CLV3 in Arabidopsis. FON4 is putative ligand of FON1, which play a role in restricting the meristem size in rice. FON4 and CLV3 are the members of CLE gene family, which encode small functional secreted peptide with a conserved 14-amino acid motif (CLE motif) near or at the C termini.
PMCID: PMC2633912  PMID: 19704753
FON4; CLAVATA; CLE; meristem; floral organ number
25.  Analyses of WOX4 transgenics provide further evidence for the evolution of the WOX gene family during the regulation of diverse stem cell functions 
Plant Signaling & Behavior  2010;5(7):916-920.
The WOX (WUSCHEL-RELATED HOMEOBOX) gene family of Arabidopsis comprises fifteen plant-specific transcriptional factors that play important development roles. Genetic, phylogenetic and genomic analyses suggest that WOX genes generally act non-autonomously to organize stem-cell and initial-cell populations within plant meristems and organ anlagen. Previous cross-complementation analyses indicate that the functional diversification of distinct WOX paralogs may be explained largely by promoter evolution, although paralog-specific protein::protein interactions are also implicated. A recent report described WOX4 function during development of the procambium, which comprises the meristematic tissues of the plant vasculature. Here we show that WOX4 fails to complement PRS1/WOX3 function, when driven from the PRS1/WOX3 native promoter. These data suggest that WOX4 identifies different DNA targets and/or interacting proteins during development of the vasculature procambium than does PRS1/WOX3 during the specification of lateral organ initial cells. The identification of super-compound leaf phenotypes induced by overexpression of the SlWOX4 ortholog in tomato suggests a functional link between vascular patterning and leaf complexity.
PMCID: PMC3014546  PMID: 20495368
tomato; Arabidopsis; WOX4; meristem; leaf; vascular tissue

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