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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.
doi:10.4161/psb.6.1.14186
PMCID: PMC3122018  PMID: 21270538
CLE; CLAVATA3-WUSCHEL; cell signaling and development; root apical meristem; arabidopsis
2.  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
3.  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.
doi:10.4161/psb.6.3.14553
PMCID: PMC3142434  PMID: 21278487
arabidopsis; CLE; embryo; polypeptide; signaling
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.
doi:10.1007/s00709-009-0095-y
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.  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.
doi:10.4161/psb.5.11.13359
PMCID: PMC3115250  PMID: 21051944
CLAVATA2; CLAVATA1; CORYNE; subcellular localization; plasma membrane; endoplasmic reticulum; receptor complex; meristem
7.  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.
doi:10.1093/pcp/pcq129
PMCID: PMC3023848  PMID: 20802224
CLV3/ESR-related (CLE); cytokinin; signal transduction; protoxylem; Arabidopsis thaliana
8.  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
9.  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.
doi:10.1007/s00425-010-1236-4
PMCID: PMC2940047  PMID: 20697738
CLE; 3D structures; Docking models; Root apical meristem; Cytokinin
10.  CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification 
SUMMARY
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.
doi:10.1111/j.1365-313X.2010.04295.x
PMCID: PMC2974754  PMID: 20626648
CLAVATA2; CLE peptide; CORYNE; CLAVATA1; receptor complex; meristem
11.  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.
doi:10.4161/psb.5.9.12477
PMCID: PMC3115079  PMID: 21490419
arabidopsis; DNA; CLEs; root morphology; plant signaling
12.  The Receptor-Like Kinase SOL2 Mediates CLE Signaling in Arabidopsis 
Plant and Cell Physiology  2008;49(11):1752-1757.
Arabidopsis sol2 mutants showed CLV3 peptide resistance. Twenty-six synthetic CLE peptides were examined in the clv1, clv2 and sol2 mutants. sol2 showed different levels of resistance to the various peptides, and the spectrum of peptide resistance was quite similar to that of clv2. SOL2 encoded a receptor-like kinase protein which is identical to CORYNE (CRN). GeneChip analysis revealed that the expression of several genes was altered in the sol2 root tip. Here, we suggest that SOL2, together with CLV2, plays an important role in the regulation of root meristem development through the CLE signaling pathway.
doi:10.1093/pcp/pcn148
PMCID: PMC2582179  PMID: 18854335
Arabidopsis thaliana; CLE peptides; CLV; Meristem; Receptor kinase; SOL2
13.  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.
doi:10.1371/journal.pgen.1002997
PMCID: PMC3499249  PMID: 23166504
14.  Peptide Signaling in Plant Development 
Current biology : CB  2011;21(9):R356-R364.
Cell-to-cell communication is integral to the evolution of multicellularity. In plant development, peptide signals relay information coordinating cell proliferation and differentiation. These peptides are often encoded by gene families and bind to corresponding families of receptors. The precise spatiotemporal expression of signals and their cognate receptors underlies developmental patterning, and expressional and biochemical changes over evolutionary time have likely contributed to the refinement and complexity of developmental programs. Here, we discuss two major plant peptide families which have central roles in plant development: the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide family and the EPIDERMAL PATTERNING FACTOR (EPF) family. We discuss how specialization has enabled the CLE peptides to modulate stem cell differentiation in various tissue types, and how differing activities of EPF peptides precisely regulate the stomatal developmental program, and we examine the contributions of these peptide families to plant development from an evolutionary perspective.
doi:10.1016/j.cub.2011.03.012
PMCID: PMC3139689  PMID: 21549958
15.  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.
doi:10.1242/dev.091314
PMCID: PMC3912870  PMID: 23578929
Arabidopsis; Cell division; Phloem; Procambium; Signalling; Vascular
16.  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.
doi:10.1371/journal.pgen.1000693
PMCID: PMC2752996  PMID: 19834537
17.  Identification of putative CLE peptide receptors involved in determinate nodulation on soybean 
Plant Signaling & Behavior  2011;6(7):1019-1023.
CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides tightly control the balance between stem cell proliferation and differentiation in several plant developmental processes. Transmission of the CLE peptide signal has been shown to be rather complex. Despite their recent identification, little is known about the receptors by which nodulation-specific CLE peptides, which were identified in soybean, are perceived. Genetic analysis has indicated that the leucine-rich repeat receptor-like kinase NARK of soybean (Glycine max) and its orthologs in other legumes are possible candidates. However, more receptors need to be identified because CLE peptides are often detected by heteromultimeric complexes. Here, we identified two additional putative CLE peptide receptor pairs in the soybean genome with a nodulation-related expression pattern, GmRLK1-GmRLK2 and GmRLK3-GmRLK4, and discuss their role in CLE peptide perception during nodulation.
doi:10.4161/psb.6.7.15575
PMCID: PMC3257784  PMID: 22004999
nodulation; soybean; receptor; LRR-RLK; CLE; peptide; determinate; AON
18.  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.
doi:10.1093/jxb/err185
PMCID: PMC3170558  PMID: 21633083
Arabidopsis; carboxypeptidase; CLE peptide; mass spectrometry; Medicago; proteomics; rice; secreted proteins; soybean; subtilisin
19.  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.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2229-13-225
PMCID: PMC3878228  PMID: 24369789
CLV3; Peptide cleavage; Flanking sequence; AA substitution; Stem cell regulation
20.  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
21.  Bioinformatic analysis of the CLE signaling peptide family 
BMC Plant Biology  2008;8:1.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2229-8-1
PMCID: PMC2254619  PMID: 18171480
22.  Mitogen-Activated Protein Kinase Regulated by the CLAVATA Receptors Contributes to Shoot Apical Meristem Homeostasis 
Plant and Cell Physiology  2010;52(1):14-29.
In Arabidopsis, the CLAVATA (CLV) pathway operates in the regulation of the size of the stem cell population in the shoot apical meristem (SAM). CLV3 functions as a small peptide ligand to negatively regulate the expression of the WUSCHEL (WUS) transcription factor through three major receptor kinase complexes of CLV1, CLV2-SUPPRESSOR OF LLP1-2 (SOL2)/CORYNE (CRN) and recently identified RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2). Aiming to understand the precise molecular details of CLV3 signaling, we investigated the contribution of phospho-signaling, potentially regulated by these kinase complexes, to the CLV pathway. We detected CLV3-triggered CLV1 phosphorylation, which is also conditioned by the rest of the CLV receptors, presumably by their direct association. Our comprehensive analysis of the activities of the respective CLV receptors on mitogen-activated protein kinases (MAPKs) suggested that the precise balanced regulation of MAPK activity by the CLV receptors is likely to be key for SAM homeostasis.
doi:10.1093/pcp/pcq157
PMCID: PMC3023851  PMID: 20965998
Arabidopsis; CLAVATA; CLE; MAPK; Nicotiana benthamiana; Shoot apical meristem
23.  Analogs of the CLV3 Peptide: Synthesis and Structure–Activity Relationships Focused on Proline Residues 
Plant and Cell Physiology  2010;52(1):30-36.
CLAVATA3 (CLV3) is a plant peptide hormone in which the proline residues are post-translationally hydroxylated and glycosylated. CLV3 plays a key role in controlling the stem cell mass in the shoot meristem of Arabidopsis thaliana. In a previous report, we identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was the smallest functional peptide derived from the CLV3 precursor. Here, we designed a series of MCLV3 analogs in which proline residues were substituted with proline derivatives or N-substituted glycines (peptoids). Peptoid substitution at Pro9 decreased bioactivity without affecting specific binding to the CLV1-related protein in cauliflower membrane. These findings suggest that peptoid-substituted peptides would be lead compounds for developing potential agonists and antagonists of CLV3.
doi:10.1093/pcp/pcq146
PMCID: PMC3023850  PMID: 20926417
Arabidopsis thaliana; CLV3; Peptide hormone; Structure–activity relationship
24.  CLE genes in plant development 
Plant Signaling & Behavior  2008;3(7):457-459.
In Arabidopsis, the CLE genes encode a family of at least 32 peptide ligands. Our gain-of-function studies demonstrated that all of the 18 genes we examined caused pleiotropic and often opposing phenotypes, including various combinations of increased root and rosette growth, root stunting, dwarfing, shoot apical meristem (SAM) arrest, asymmetric leaf development, and “shrublike” phenotypes. Many CLE genes caused similar phenotypes that correlated with common amino acid substitutions among subsets of the genes, suggesting key amino acids necessary for certain phenotypes. The pleiotropic phenotypes we observed were the results of integrated hypermorphic and global neomorphic responses to abundant ectopic ligands through multiple signaling pathways. The phenotypes are also suggestive of wide ranging, often antagonistic roles played by these genes in plant development. The interpretations of our findings and some apparently contradictory recent results are discussed in this context.
PMCID: PMC2634428  PMID: 19704484
arabidopsis; clavata; CLE; hypermorphy; neomorphy; peptide ligand; plant development
25.  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.
doi:10.1093/jxb/ert008
PMCID: PMC3617822  PMID: 23386683
Autoregulation of nodulation; CLE peptides; legumes; nodulation; soybean; symbiosis.

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