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CLE proteins are a class of signalling factors involved in plant growth and abiotic stress response. They play crucial roles in processes such as cell differentiation, chlorophyll synthesis and abscisic acid (ABA) signal transduction. However, the function of the CLE genes in Sorghum bicolor remains unclear. In this study, 42 sorghum CLE genes were identified, and their evolutionary relationship, gene structure, amino acid sequence and homologous genes were analysed. We also examined the expression levels of CLE genes under various treatment conditions. Transcriptome data showed that there were significant differences in the expression patterns of 42 CLE genes in different tissues and organs. It is worth noting that SbCLE39 is mainly highly expressed in sorghum roots. At the same time, the expression of SbCLE39 decreased significantly under salt and ABA treatment. Compared with wild‐type yeast cells (EV), yeast cells with high expression of SbCLE39 had lower tolerance to salt stress. In addition, the excessive accumulation of ABA caused by external application of SbCLE39p reduced the salt tolerance of sorghum. These findings suggest that SbCLE39 negatively regulates the salt tolerance of sorghum. These results lay a foundation for revealing the mechanism of CLE genes regulating the salt tolerance of sorghum and are of great significance for the cultivation of salt‐tolerant crops. [ABSTRACT FROM AUTHOR]

SUMMARY: Symbiotic nitrogen fixation is an energy‐intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR‐Cas9‐mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate‐tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate‐triggered inhibition of nodulation, as the expression of nitrate‐responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O‐arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4‐GmNIC1a/b module in mediating high nitrate‐induced inhibition of nodulation. Significance Statement: This study highlights the crucial role of the GmNLP1/4‐GmNIC1a/b module in mediating high nitrate‐induced inhibition of nodulation. [ABSTRACT FROM AUTHOR]

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced. [ABSTRACT FROM AUTHOR]

In potato, phloem tissues transport sugars and signal molecules to the tuber for growth and storage. The CLAVATA3/ESR-like (CLE) family of plant peptides plays an important role in regulating plant development. In this study, we identified a set of phloem-expressed CLE genes in Solanum tuberosum L. (StCLEs). We analyzed the phloem transcriptome of potato and found that 10 out of 41 StCLE genes were expressed in phloem cells, with StCLE12 and StCLE19 showing the highest expression levels. StCLE12 has an identical CLE domain to the Arabidopsis TDIF peptides, which are known to play a crucial role in maintaining the vascular meristem. StCLE19 has the highest sequence similarity to the Arabidopsis CLE25 peptide, which is involved in the formation of the phloem element and signaling in response to dehydration stress. The overexpression of StCLE12 and another potato TDIF-like gene, StCLE8, promoted vascular cell proliferation and delayed leaf senescence. On the other hand, plants with overexpression of StCLE19 were unable to form adventitious roots and demonstrated the absence of ordered cambium cell layers in the vascular bundles. [ABSTRACT FROM AUTHOR]

CLE peptides are well-known hormonal regulators of plant development, but their role in somatic embryogenesis remains undetermined. CLE genes are often regulated by WOX transcription factors and, in their turn, regulate the expression level of WOX genes. In this study, we used in vitro cultivation, as well as qPCR and transcriptomic analysis, to find CLE peptides which could regulate the MtWOX9-1 gene, stimulating somatic embryogenesis in Medicago truncatula. Three CLE peptides were found which could probably be such regulators, but none of them was found to influence MtWOX9-1 expression in the embryogenic calli. Nevertheless, overexpression of one of CLE genes under study, MtCLE16, decreased somatic embryogenesis intensity. Additionally, overexpression of MtCLE08 was found to suppress expression of MtWOX13a, a supposed antagonist of somatic embryo development. Our findings can be helpful for the search for new regeneration regulators which could be used for plant transformation. [ABSTRACT FROM AUTHOR]

In potato, phloem tissues transport sugars and signal molecules to the tuber for growth and storage. The CLAVATA3/ESR-like (CLE) family of plant peptides plays an important role in regulating plant development. In this study, we identified a set of phloem-expressed CLE genes in Solanum tuberosum L. (StCLEs). We analyzed the phloem transcriptome of potato and found that 10 out of 41 StCLE genes were expressed in phloem cells, with StCLE12 and StCLE19 showing the highest expression levels. StCLE12 has an identical CLE domain to the Arabidopsis TDIF peptides, which are known to play a crucial role in maintaining the vascular meristem. StCLE19 has the highest sequence similarity to the Arabidopsis CLE25 peptide, which is involved in the formation of the phloem element and signaling in response to dehydration stress. The overexpression of StCLE12 and another potato TDIF-like gene, StCLE8, promoted vascular cell proliferation and delayed leaf senescence. On the other hand, plants with overexpression of StCLE19 were unable to form adventitious roots and demonstrated the absence of ordered cambium cell layers in the vascular bundles.

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

Over time, plants have evolved flexible self-organizing patterning mechanisms to adapt tissue functionality for continuous organ growth. An example of this process is the multicellular organization of cells into a vascular network in foliar organs. An important, yet poorly understood component of this process is secondary vein branching, a mechanism employed to extend vascular tissues throughout the cotyledon surface. Here, we uncover two distinct branching mechanisms during embryogenesis by analyzing the discontinuous vein network of the double mutant cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Similar to wild-type embryos, distal veins in cvp2 cvl1 embryos arise from the bifurcation of cell files contained in the midvein, whereas proximal branching is absent in this mutant. Restoration of this process can be achieved by increasing OCTOPUS dosage as well as by silencing RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) expression. Although RPK2-dependent rescue of cvp2 cvl1 is auxin- and CLE peptide-independent, distal branching involves polar auxin transport and follows a distinct regulatory mechanism. Our work defines a genetic network that confers plasticity to Arabidopsis embryos to spatially adapt vascular tissues to organ growth. [ABSTRACT FROM AUTHOR]

Abstract Background Signal peptides are essential for plant growth and development. In plants, biological processes including cell-cell communication, cellular proliferation and differentiation, cellular determination of self-incompatibility, and defensive responses, all depend heavily on peptide-signaling networks such as CLE (CLAVATA3/Embryo surrounding region-related). The CLEs are indispensable in different periods of plant growth and development, especially in maintaining the balance between proliferation and differentiation of stem cells in various meristematic tissues. The working system of CLE genes in cucumber, an important economical vegetable (Cucumis sativus L.), has not been fully studied yet. The distributional patterns of chromosome-level genome assembly in cucumber provide a fundamental basis for a genome-wide comparative analysis of CLE genes in such plants. Results A total of 26 individual CLE genes were identified in Chinese long ‘9930’ cucumber, the majority of which belong to unstable short alkaline and hydrophilic peptides. A comparative analysis showed a close relationship in the development of CLE genes among Arabidopsis thaliana, melon, and cucumber. Half of the exon-intron structures of all CsCLEs genes are single-exon genes, and motif 1, a typical CLE domain near the C-terminal functioning in signal pathways, is found in all cucumber CLE proteins but CsCLE9. The analysis of CREs (Cis-Regulatory Elements) in the upstream region of the 26 cucumber CLE genes indicates a possible relationship between CsCLE genes and certain functions of hormone response elements. Cucumber resulted closely related to Arabidopsis and melon, having seven and 15 orthologous CLE genes in Arabidopsis and melon, respectively. Additionally, the calculative analysis of a pair of orthologous genes in cucumber showed that as a part of the evolutionary process, CLE genes are undergoing a positive selection process which leads to functional differentiation. The specific expression of these genes was vigorous at the growth and development period and tissues. Cucumber gene CLV3 was overexpressed in Arabidopsis, more than half of the transformed plants in T1 generation showed the phenomena of obvious weakness of the development of growing point, no bolting, and a decreased ability of plant growth. Only two bolted strains showed that either the pod did not develop or the pod was short, and its development was significantly inferior to that in the wild type. Conclusions In this study, 26 CLE genes were identified in Chinese long ‘9930’ cucumber genome. The CLE genes were mainly composed of alkaline hydrophilic unstable proteins. The genes of the CLE family were divided into seven classes, and shared close relationships with their homologs in Arabidopsis and melon. The specific expression of these genes was evaluated in different periods of growth and tissue development, and CLV3, which the representative gene of the family, was overexpressed in Arabidopsis, suggesting that it has a role in bolting and fruit bearing in cucumber.

Legume plants form nitrogen-fixing nodules on their roots in symbiosis with soil bacteria rhizobia. The number of symbiotic nodules is controlled by a host plant via a systemic mechanism known as autoregulation of nodulation (AON). The key players of AON are the CLE peptides which are produced in the root in response to rhizobia inoculation and are transported via xylem to the shoot. In the shoot, the CLE peptides are recognized by a CLV1-like receptor kinase, which results in subsequent inhibition of nodule development in the root via a negative feedback mechanism. In addition to the CLE peptides induced by rhizobia, nitrate-induced CLE peptides involved in the control of nodulation have been identified. In Medicago truncatula, the MtCLE34 gene has been described, which was activated by nitrate and in response to rhizobial inoculation. However, this gene contains a premature stop codon in the reference M. truncatula genome of the A17 line, and therefore, it was suggested to be a pseudogene. Here, we analyzed nucleotide sequences of the MtCLE34 gene available from the genomes of different M. truncatula accessions from the Medicago HAPMAP project and found that the majority of M. truncatula accession lines do not carry nonsense mutations in the MtCLE34 gene and should encode functional products. Overexpression of the MtCLE34 gene from the R108 line, which does not have a premature stop codon, did not inhibit nodulation. Therefore, in spite of having high sequence similarity to the nodulation-suppressing CLE genes, the MtCLE34 gene from the R108 line was not able to trigger AON in M. truncatula. Our findings shed light on the evolutionary changes in the CLE proteins in legume plants and can be used in the future to understand which amino acid residues within CLE proteins could be important for their ability to suppress nodulation. [ABSTRACT FROM AUTHOR]

Main conclusion: Plant CLE peptides, which regulate stem cell maintenance in shoot and root meristems and in vascular bundles through LRR family receptor kinases, are novel, complex, and to some extent conserved. Over the past two decades, peptide ligands of the CLAVATA3 (CLV3) /Embryo Surrounding Region (CLE) family have been recognized as critical short- and long-distance communication signals in plants, especially for stem cell homeostasis, cell fate determination and physiological responses. Stem cells located at the shoot apical meristem (SAM), the root apical meristem (RAM) and the procambium divide and differentiate into specialized cells that form a variety of tissues such as epidermis, ground tissues, xylem and phloem. In the SAM of Arabidopsis (Arabidopsis thaliana), the CLV3 peptide restricts the number of stem cells via leucine-rich repeat (LRR)-type receptor kinases. In the RAM, root-active CLE peptides are critical negative regulators, while ROOT GROWTH FACTOR (RGF) peptides are positive regulators in stem cell maintenance. Among those root-active CLE peptides, CLE25 promotes, while CLE45 inhibits phloem differentiation. In vascular bundles, TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/CLE41/CLE44 promotes procambium cell division, and prevents xylem differentiation. Orthologs of CLV3 have been identified in liverwort (Marchantia polymorpha), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays) and lotus (Lotus japonicas), suggesting that CLV3 is an evolutionarily conserved signal in stem cell maintenance. However, functional characterization of endogenous CLE peptides and corresponding receptor kinases, and the downstream signal transduction has been challenging due to their genome-wide redundancies and rapid evolution. [ABSTRACT FROM AUTHOR]

CLE peptides are well-known hormonal regulators of plant development, but their role in somatic embryogenesis remains undetermined. CLE genes are often regulated by WOX transcription factors and, in their turn, regulate the expression level of WOX genes. In this study, we used in vitro cultivation, as well as qPCR and transcriptomic analysis, to find CLE peptides which could regulate the MtWOX9-1 gene, stimulating somatic embryogenesis in Medicago truncatula. Three CLE peptides were found which could probably be such regulators, but none of them was found to influence MtWOX9-1 expression in the embryogenic calli. Nevertheless, overexpression of one of CLE genes under study, MtCLE16, decreased somatic embryogenesis intensity. Additionally, overexpression of MtCLE08 was found to suppress expression of MtWOX13a, a supposed antagonist of somatic embryo development. Our findings can be helpful for the search for new regeneration regulators which could be used for plant transformation.

The receptor-like kinases (RLKs) CLAVATA1 (CLV1) and BARELY ANY MERISTEMs (BAM1–BAM3) form the CLV1 family (CLV1f), which perceives peptides of the CLV3/EMBRYO SURROUNDING REGION (ESR)-related (CLE) family within various signaling pathways of Arabidopsis thaliana. CLE peptide signaling, which is required for meristem size control, vascular development, and pathogen responses, involves the formation of receptor complexes at the plasma membrane. These complexes comprise RLKs and co-receptors in varying compositions depending on the signaling context, and regulate expression of target genes, such as WUSCHEL (WUS). How the CLE signal is transmitted intracellularly after perception at the plasma membrane is not known in detail. Here, we found that the membrane-associated receptor-like cytoplasmic kinase (RLCK) MAZZA (MAZ) and additional members of the Pti1-like protein family interact in vivo with CLV1f receptors. MAZ, which is widely expressed throughout the plant, localizes to the plasma membrane via post-translational palmitoylation, potentially enabling stimulus-triggered protein re-localization. We identified a role for a CLV1–MAZ signaling module during stomatal and root development, and redundancy could potentially mask other phenotypes of maz mutants. We propose that MAZ, and related RLCKs, mediate CLV1f signaling in a variety of developmental contexts, paving the way towards understanding the intracellular processes after CLE peptide perception. [ABSTRACT FROM AUTHOR]

Background: Signal peptides are essential for plant growth and development. In plants, biological processes including cell-cell communication, cellular proliferation and differentiation, cellular determination of self-incompatibility, and defensive responses, all depend heavily on peptide-signaling networks such as CLE (CLAVATA3/Embryo surrounding region-related). The CLEs are indispensable in different periods of plant growth and development, especially in maintaining the balance between proliferation and differentiation of stem cells in various meristematic tissues. The working system of CLE genes in cucumber, an important economical vegetable (Cucumis sativus L.), has not been fully studied yet. The distributional patterns of chromosome-level genome assembly in cucumber provide a fundamental basis for a genome-wide comparative analysis of CLE genes in such plants. Results: A total of 26 individual CLE genes were identified in Chinese long '9930' cucumber, the majority of which belong to unstable short alkaline and hydrophilic peptides. A comparative analysis showed a close relationship in the development of CLE genes among Arabidopsis thaliana, melon, and cucumber. Half of the exon-intron structures of all CsCLEs genes are single-exon genes, and motif 1, a typical CLE domain near the C-terminal functioning in signal pathways, is found in all cucumber CLE proteins but CsCLE9. The analysis of CREs (Cis-Regulatory Elements) in the upstream region of the 26 cucumber CLE genes indicates a possible relationship between CsCLE genes and certain functions of hormone response elements. Cucumber resulted closely related to Arabidopsis and melon, having seven and 15 orthologous CLE genes in Arabidopsis and melon, respectively. Additionally, the calculative analysis of a pair of orthologous genes in cucumber showed that as a part of the evolutionary process, CLE genes are undergoing a positive selection process which leads to functional differentiation. The specific expression of these genes was vigorous at the growth and development period and tissues. Cucumber gene CLV3 was overexpressed in Arabidopsis, more than half of the transformed plants in T1 generation showed the phenomena of obvious weakness of the development of growing point, no bolting, and a decreased ability of plant growth. Only two bolted strains showed that either the pod did not develop or the pod was short, and its development was significantly inferior to that in the wild type. Conclusions: In this study, 26 CLE genes were identified in Chinese long '9930' cucumber genome. The CLE genes were mainly composed of alkaline hydrophilic unstable proteins. The genes of the CLE family were divided into seven classes, and shared close relationships with their homologs in Arabidopsis and melon. The specific expression of these genes was evaluated in different periods of growth and tissue development, and CLV3, which the representative gene of the family, was overexpressed in Arabidopsis, suggesting that it has a role in bolting and fruit bearing in cucumber. [ABSTRACT FROM AUTHOR]

Legume plants form nitrogen-fixing nodules on their roots in symbiosis with soil bacteria rhizobia. The number of symbiotic nodules is controlled by a host plant via a systemic mechanism known as autoregulation of nodulation (AON). The key players of AON are the CLE peptides which are produced in the root in response to rhizobia inoculation and are transported via xylem to the shoot. In the shoot, the CLE peptides are recognized by a CLV1-like receptor kinase, which results in subsequent inhibition of nodule development in the root via a negative feedback mechanism. In addition to the CLE peptides induced by rhizobia, nitrate-induced CLE peptides involved in the control of nodulation have been identified. In Medicago truncatula, the MtCLE34 gene has been described, which was activated by nitrate and in response to rhizobial inoculation. However, this gene contains a premature stop codon in the reference M. truncatula genome of the A17 line, and therefore, it was suggested to be a pseudogene. Here, we analyzed nucleotide sequences of the MtCLE34 gene available from the genomes of different M. truncatula accessions from the Medicago HAPMAP project and found that the majority of M. truncatula accession lines do not carry nonsense mutations in the MtCLE34 gene and should encode functional products. Overexpression of the MtCLE34 gene from the R108 line, which does not have a premature stop codon, did not inhibit nodulation. Therefore, in spite of having high sequence similarity to the nodulation-suppressing CLE genes, the MtCLE34 gene from the R108 line was not able to trigger AON in M. truncatula. Our findings shed light on the evolutionary changes in the CLE proteins in legume plants and can be used in the future to understand which amino acid residues within CLE proteins could be important for their ability to suppress nodulation.

Summary: Flowering plants consist of highly differentiated organs, including roots, leaves, shoots and flowers, which have specific roles: root system for water and nutrient uptake, leaves for photosynthesis and gas exchange and reproductive organs for seed production. The communication between organs through the vascular system, by which water, nutrient and signaling molecules are transported, is essential for coordinated growth and development of the whole plant, particularly under adverse conditions. Here, we highlight recent progress in understanding how signaling pathways of plant hormones are associated with long‐distance stress and developmental signals, with particular focus on environmental stress responses. In addition to the root‐to‐shoot peptide signal that induces abscisic acid accumulation in leaves under drought stress conditions, we summarize the diverse stress‐responsive peptide signals reported to date to play a role in environmental responses. Significance Statement: Among the increasing number of stress‐responsive peptides identified, CLE25 synthesized in response to soil drought enables plants to biosynthesize abscisic acid rapidly in leaves and reduce water consumption by stomatal closure. Here recent findings of long‐distance stress and developmental signals are summarized and their relationships with other environmental stress responses are discussed. [ABSTRACT FROM AUTHOR]

Plants and arbuscular mycorrhizal fungi (AMF) engage in mutually beneficial symbioses based on a reciprocal exchange of nutrients. The beneficial character of the symbiosis is maintained through a mechanism called autoregulation of mycorrhization (AOM). AOM includes root-to-shoot-to-root signaling; however, the molecular details of AOM are poorly understood. AOM shares many features of autoregulation of nodulation (AON) where several genes are known, including the receptor-like kinase SUPER NUMERIC NODULES (SUNN), root-to-shoot mobile CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) peptides, and the hydroxyproline O -arabinosyltransferase ROOT DETERMINED NODULATION1 (RDN1) required for post-translational peptide modification. In this work, CLE53 was identified to negatively regulate AMF symbiosis in a SUNN- and RDN1-dependent manner. CLE53 expression was repressed at low phosphorus, while it was induced by AMF colonization and high phosphorus. CLE53 overexpression reduced AMF colonization in a SUNN- and RDN1 dependent manner, while cle53 , rdn1 , and sunn mutants were more colonized than the wild type. RNA-sequencing identified 700 genes with SUNN-dependent regulation in AMF-colonized plants, providing a resource for future identification of additional AOM genes. Disruption of AOM genes in crops potentially constitutes a novel route for improving AMF-derived phosphorus uptake in agricultural systems with high phosphorus levels. [ABSTRACT FROM AUTHOR]

Summary: The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)‐RELATED (CLE) peptide ligands in connection with their receptors are important players in cell‐to‐cell communications in plants. Here, we investigated the function of the Populus CLV3/ESR‐RELATED 47 (PttCLE47) gene during secondary growth and wood formation in hybrid aspen (Populus tremula × tremuloides) using an RNA interference (RNAi) approach.Expression of PttCLE47 peaks in the vascular cambium. Silencing of the PttCLE47 gene expression affected lateral expansion of stems and decreased apical height growth and leaf size.In particular, PttCLE47 RNAi trees exhibited a narrower secondary xylem zone with less xylem cells/cell file. The reduced radial growth phenotype also correlated with a reduced number of cambial cell layers. In agreement with these results, expression of several cambial regulator genes was downregulated in the stems of the transgenic trees in comparison with controls.Altogether, these results suggest that the PttCLE47 gene is a major positive regulator of cambial activity in hybrid aspen, mainly promoting the production of secondary xylem. Furthermore, in contrast to previously characterized CLE genes expressed in the wood‐forming zone, PttCLE47 appears to be active at its site of expression. [ABSTRACT FROM AUTHOR]

Receptors, in particular receptor kinases, are an integral part of the signaling pathways that regulate plant growth, development, and response to external factors. The receptors of CLE peptides involved in the development and functioning of various types of meristems are a group of kinases, most of which have leucine-rich repeats in the ligand-binding domain. Long-term studies of receptor kinases involved in signaling from CLE peptides are reviewed. [ABSTRACT FROM AUTHOR]

Summary: The arbuscular mycorrhizal (AM) symbiosis is a beneficial association established between land plants and the members of a subphylum of fungi, the Glomeromycotina. How the two symbiotic partners regulate their association is still enigmatic. Secreted fungal peptides are candidates for regulating this interaction.We searched for fungal peptides with similarities with known plant signalling peptides.We identified CLAVATA (CLV)/EMBRYO SURROUNDING REGION (ESR)‐RELATED PROTEIN (CLE) genes in phylogenetically distant AM fungi: four Rhizophagus species and one Gigaspora species. These CLE genes encode a signal peptide for secretion and the conserved CLE C‐terminal motif. They seem to be absent in the other fungal clades. Rhizophagus irregularis and Gigaspora rosea CLE genes (RiCLE1 and GrCLE1) are transcriptionally induced in symbiotic vs asymbiotic conditions. Exogenous application of synthetic RiCLE1 peptide on Medicago truncatula affects root architecture, by slowing the apical growth of primary roots and stimulating the formation of lateral roots. In addition, pretreatment of seedlings with RiCLE1 peptide stimulates mycorrhization.Our findings demonstrate for the first time that in addition to plants and nematodes, AM fungi also possess CLE genes. These results pave the way for deciphering new mechanisms by which AM fungi modulate plant cellular responses during the establishment of AM symbiosis. [ABSTRACT FROM AUTHOR]

CLE peptides (CLAVATA3/ENDOSPERM SURROUNDING REGION) are signal molecules or plant peptide hormones that play an important role in regulation of development of various meristems governing the expression of WOX (WUSCHEL-RELATED HOMEOBOX) genes. In particular, CLE peptides belonging to a small TDIF (Tracheary Element Differentiation Inhibitory Factor) group are responsible for the operation of gene WOX4 controlling the development of cambium and the conducting system. We looked into the role of CLE41 peptide from the TDIF group in the development of storage root in two species of the genus Raphanus: cultivated radish (Raphanus sativus var. radicula Pers.) that is a popular root crop with a storage root and its ancestor wild radish (Raphanus raphanistrum L.) where storage parenchyma of the root is poorly developed. It was shown that overexpression of gene RsCLE41 and plant treatment with exogenous peptide CLE41 influenced the development of cambium and xylem in the roots of R. sativus and R. raphanistrum and affected expression of the genes from different groups. One could say that peptide CLE41 activates expression of the genes whose homologues in arabidopsis play a key role in the maintenance of cambium (RsWOX4, RsWOX14, RsHAM4, and RsCYCD3). In the storage root of radish, peptide CLE41 activates proliferation of cambium cells reducing the amount of one of the xylem’s elements (lignified parenchyma). The obtained results point to an important role of CLE41 in the development of storage root in radish. [ABSTRACT FROM AUTHOR]

The ligand-receptor-mediated intercellular communication system plays important roles in coordinating developmental and physiological events in multicellular organisms. In plants, CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides and their cognate receptors are thought to be involved in various aspects of the plant life cycle. Although the importance of this communication is broadly recognized, most CLE peptides are yet to be functionally characterized. A major problem in research on small signaling peptide-encoding genes is the limited number of loss-of-function mutants available due to their small gene size. CRISPR/Cas9-mediated gene targeting has the potential to overcome this problem, as it can be used to generate targeted mutations in essentially any gene, regardless of size. Here we generated a series of mutants of CLE-peptide-encoding genes. Newly generated clv3 and cle40 mutants reproduced the expected mutant phenotypes in the shoot apical meristem and root meristem, respectively. Our results show that CRISPR/Cas9-mediated gene targeting is a powerful tool for genetic analyses, even of small genes. We also report a novel mutant for CLE44 [which is thought to encode a tracheary elements differentiation inhibitory factor (TDIF)] and show that CLE44 contributes to vascular development. The bioresources presented here will be a powerful tool for further characterization of CLE peptides. [ABSTRACT FROM AUTHOR]

Stem cells (SCs) are undifferentiated cells of multicellular organisms that can divide, self-renew, and differentiate. Despite the differences in their properties, the general principles of the existence of SCs can be distinguished in all multicellular organisms. In plants, SCs are found in meristems, structures that ensure the continuous growth of a plant and provide material for the formation of various specialized tissues. There are numerous types of meristems: shoot apical meristem (SAM) and root apical meristem (RAM), and lateral meristems (LMs) (procambium, cambium, and pericycle), as well as the so-called irregular meristems, developing under certain conditions (callus, meristems of symbiotic nodules, spontaneous and pathogen-induced tumors, etc.). Specific mechanisms of regulation, which are based on the interaction of plant hormones and the major groups of transcription factors, were identified for each meristem. The activity of meristems is determined by two opposing processes: proliferation and self-renewal of SCs in the central part of the meristem and differentiation of specialized cells in the periphery. WOX-CLAVATA systems are a regulatory component conservative for different meristems, which ensures the consistency of the composition of the meristem, as well as the balance of SC proliferation and differentiation. In this review, we consider the similarities and differences between the principles of the organization of SC niches in plants and animals, as well as in a variety of meristems of higher plants; special attention will be paid to the role of WOX-CLAVATA systems in maintaining meristems and their interaction with other meristem regulators. [ABSTRACT FROM AUTHOR]

Peptide signals mediate a variety of cell-to-cell communication crucial for plant growth and development. During Arabidopsis thaliana vascular development, a CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) family peptide hormone, TDIF (tracheary element differentiation inhibitory factor), regulates procambial cell fate by its inhibitory activity on xylem differentiation. To address if this activity is conserved among vascular plants, we performed comparative analyses of TDIF signaling in non-flowering vascular plants (gymnosperms, monilophytes and lycophytes). We identified orthologs of TDIF/CLE as well as its receptor TDR/PXY (TDIF RECEPTOR/PHLOEM INTERCALATED WITH XYLEM) in Ginkgo biloba, Adiantum aethiopicum and Selaginella kraussiana by RACE-PCR. The predicted TDIF peptide sequences in seed plants and monilophytes were identical to that of A. thaliana TDIF. We examined the effects of exogenous CLE peptide-motif sequences of TDIF in these species. We found that liquid culturing of dissected leaves or shoots was useful for examining TDIF activity during vascular development. TDIF treatment suppressed xylem/tracheary element differentiation of procambial cells in G. bioloba and A. aethiopicum leaves. In contrast, neither TDIF nor putative endogenous TDIF inhibited xylem differentiation in developing shoots and rhizophores of S. kraussiana. These data suggest that activity of TDIF in vascular development is conserved among extant euphyllophytes. In addition to the conserved function, via liquid culturing of its bulbils, we found a novel inhibitory activity on root growth in the monilophyte Asplenium x lucrosum suggesting lineage-specific co-option of peptide signaling occurred during the evolution of vascular plant organs.

A fundamental aspect of plant development is the coordination of growth through endogenous signals and its integration with environmental inputs. Similar to animals, plants frequently use cell surface-localized receptors to monitor such stimuli, for instance through plasma membrane-integral receptor-like kinases (RLKs). Compared to other organisms, plants possess a large number of RLKs (more than 600 in Arabidopsis thaliana), which implies that ligand-receptor-mediated molecular mechanisms regulate a wide range of processes during plant development. Here, we focus on A. thaliana RLKs of the CLAVATA 1 (CLV1) type, which orchestrate key steps during plant development, including the regulation of meristem maintenance, anther development, vascular tissue formation, and root system architecture. These receptors are regulated by small signalling peptides that belong to the family of CLE (CLV3 / EMBRYO SURROUNDING REGION) ligands. We discuss different aspects of plant development that are regulated by these receptors in light of their molecular mechanism of action. As so often, the intensive research on this group of plant RLKs has raised many intriguing questions, which remain to be answered. [ABSTRACT FROM AUTHOR]

The remarkable plasticity of post-embryonic plant development is due to groups of stem-cell-containing structures called meristems. In the shoot, meristems continuously produce organs such as leaves, flowers, and stems. Nearly two decades ago the WUSCHEL/CLAVATA (WUS/CLV) negative feedback loop was established as being essential for regulating the size of shoot meristems by maintaining a delicate balance between stem cell proliferation and cell recruitment for the differentiation of lateral primordia. Recent research in various model species ( Arabidopsis , tomato, maize, and rice) has led to discoveries of additional components that further refine and improve the current model of meristem regulation, adding new complexity to a vital network for plant growth and productivity. [ABSTRACT FROM AUTHOR]

Background: Radish (Raphanus sativus L.) is a widespread agricultural plant forming storage root due to extensive secondary growth which involves cambium proliferation and differentiation of secondary conductive tissues. Closely related to the model object Arabidopsis thaliana, radish is a suitable model for studying processes of secondary growth and storage root development. CLE peptides are a group of peptide phytohormones which play important role in the regulation of primary meristems such as SAM, RAM, and procambium, as well as secondary meristems. However, the role of CLE peptides in lateral growth of root during storage root formation has not been studied to date. Results: In present work we studied the role of CLE peptides in the development of storage root in radish. We have identified 18 CLE genes of radish (RsCLEs) and measured their expression in various plant organs and also at different stages of root development in R. sativus and Raphanus raphanistrum--its close relative which does not form storage root. We observed significant decline of expression levels for genes RsCLE1, 2, 11, 13, and 16, and also multifold increase of expression levels for genes RsCLE19, and 41 during secondary root growth in R. sativus but not in R. raphanistrum. Expression of RsCLE 2, 19, and 41 in R. sativus root was confined to certain types of tissues while RsCLE1, 11, 13, and 16 expressed throughout the root. Experiments on overexpression of RsCLE2, 19 and 41 or treatment of radish plants with synthetic CLE peptides revealed that CLE19 and CLE2 increase the number of xylem elements, and CLE41 induces the formation of extra cambium foci in secondary xylem. Expression levels of RsCLE2 and 19 strongly decrease in response to exogenous cytokinin, while auxin causes dramatic increase of RsCLE19 expression level and decrease of RsCLE41 expression. Conclusions: Our data allow us to hypothesize about the role of RsCLE2, 19 and 41 genes in the development of storage root of Raphanus sativus, e.g. RsCLE19 may play a role in auxin-dependent processes of xylem differentiation and RsCLE41 stimulates cambium activity. [ABSTRACT FROM AUTHOR]

Lateral meristems (pericycle, procambium and cambium, phellogen) are positioned in parallel to the lateral surface of the organ, where they are present, and produce concentric layers of undifferentiated cells. Primary lateral meristems, procambium and pericycle, arise during embryogenesis; secondary lateral meristems, cambium and phellogen, - during post embryonic development. Pericycle is most pluripotent plant meristem, as it may give rise to a variety of other types of meristems: lateral meristems (cambium, phellogen), apical meristems of lateral roots, and also shoot meristems during plant in vitro regeneration. Procambium and cambium developing from it give rise to the vascular tissues of the stems and roots, ensuring their thickening. The review considers the genetic control of lateral meristem development and the role of phytohormones in the control of their activities. [ABSTRACT FROM AUTHOR]

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 GmNICI (nitrate-induced and acting locally) and GmRICI (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 GmRICI. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRICI 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 GmRICI and GmNICI 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 GmRICI significantly enhanced activity of GmNICI. In contrast, the comparable GmNICI domains reduced the NSA of GmRICI. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation. [ABSTRACT FROM AUTHOR]

CLE ( CLAVATA3/ ENDOSPERM SURROUNDING REGION) peptides are peptide phytohormones playing the important role in the regulation of various type meristem development and also in the interaction between plants and parasites and symbionts. At the same time, the interaction of CLE peptides with other phytohormones, participating in these processes, is essentially unstudied. Using real-time RT-PCR, we analyzed expression of some genes encoding CLE peptides in radish ( Raphanus sativus var. radicula Pers.) under normal conditions and after treatment with the cytokinin 6-benzyladenine. Even after short-term (30 min) action of cytokinin, expression of group A CLE genes was manifold suppressed, whereas cytokinin did not affect expression of group B CLE genes. Radish lines contrasting in the trait 'spontaneous tumor formation' demonstrated similar dynamics of CLE gene expression in response to cytokinin treatment but differed in the levels of their expression. A possibility of interaction between CLE peptides and cytokinins in the spontaneous development of tumors in radish lines is discussed. The important part of the work was the selection of optimal reference genes for the analysis of radish gene expression by real-time RT-PCR method. Most stable expression was observed for the genes of ubiquitin ( RsUBQ) and glyceraldehydes-3-phosphate dehydrogenase ( RsGAPDH). [ABSTRACT FROM AUTHOR]

Peptide signaling presumably occupies a central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. Here we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/ENDOSPERM SURROUNDING REGION 45 (CLE45) peptide ligand. WT plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a WT phenotype, however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in WT, but not in bam3 mutants. BAM3, BRX, and CLE45 are expressed in a similar spatiotemporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems, and laser ablation experiments suggest that intertwined regulatory activity of BRX, BAM3, and CLE45 could be involved in the proper transition of protophloem cells from proliferation to differentiation, thereby impinging on postembryonic growth capacity of the root meristem. [ABSTRACT FROM AUTHOR]

Meristems are morphogenic plant tissues comprising the pool of stem cells initiating specialized tissues. The balance of stem cell proliferation and differentiation depends on antagonistic action of genes WOX encoding homeodomain-containing transcription factors and CLAVATA-like systems controlling their expression. The CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) peptides and their receptor protein kinases comprise the basis of the CLAVATA-like systems. Definite member of the family of CLE peptides (CLV3 in the shoot, CLE40 in the root, and CLE41/CLE44 in the procambium) are central regulators of primary meristem development. The role of CLE peptides in the development of secondary meristems, such as nodule meristems of legumes, is studied. CLE peptides are found also outside the plant kingdom, e.g., parasitic nematodes. The review considers CLE peptides as universal regulators of meristem development. [ABSTRACT FROM AUTHOR]

Oligopeptide transporters (OPTs) are found in fungi, bacteria and plants. The nine Arabidopsis thaliana OPT genes are expressed mainly in the vasculature and are thought to transport tetra- and pentapeptides, and peptide-like substrates such as glutathione. Expression of AtOPT6 in Xenopus laevis oocytes demonstrated that AtOPT6 transports many tetra- and pentapeptides. In addition, AtOPT6 transported reduced glutathione (GSH), a tripeptide, but not oxidized glutathione (GSSG). Recent data showed that Candida albicans OPTs can transport peptides up to eight amino acids in length. AtOPT6 transported mammalian signaling peptides up to 10 amino acids in length and, in addition, known plant development- and nematode pathogenesis-associated peptides up to 13 amino acids long. AtOPT6 displayed high affinity for penta- and dodecapeptides, but low affinity for GSH. In comparison the Saccharomyces cerevisiae ScOPT1 was incapable of transporting any of the longer peptides tested. These data demonstrate the necessity of experimentally determining substrate specificity of individual OPTs, and lay a foundation for structure/function studies. Characterization of the AtOPT6 substrate range provides a basis for investigating the possible physiological function of AtOPT6 in peptide signaling and thiol transport in response to stress. [ABSTRACT FROM PUBLISHER]

Abstract: The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure–activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1. [Copyright &y& Elsevier]

Main Conclusion: Plant CLE peptides, which regulate stem cell maintenance in shoot and root meristems and in vascular bundles through LRR family receptor kinases, are novel, complex, and to some extent conserved. Over the past two decades, peptide ligands of the CLAVATA3 (CLV3) /Embryo Surrounding Region (CLE) family have been recognized as critical short- and long-distance communication signals in plants, especially for stem cell homeostasis, cell fate determination and physiological responses. Stem cells located at the shoot apical meristem (SAM), the root apical meristem (RAM) and the procambium divide and differentiate into specialized cells that form a variety of tissues such as epidermis, ground tissues, xylem and phloem. In the SAM of Arabidopsis (Arabidopsis thaliana), the CLV3 peptide restricts the number of stem cells via leucine-rich repeat (LRR)-type receptor kinases. In the RAM, root-active CLE peptides are critical negative regulators, while ROOT GROWTH FACTOR (RGF) peptides are positive regulators in stem cell maintenance. Among those root-active CLE peptides, CLE25 promotes, while CLE45 inhibits phloem differentiation. In vascular bundles, TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/CLE41/CLE44 promotes procambium cell division, and prevents xylem differentiation. Orthologs of CLV3 have been identified in liverwort (Marchantia polymorpha), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays) and lotus (Lotus japonicas), suggesting that CLV3 is an evolutionarily conserved signal in stem cell maintenance. However, functional characterization of endogenous CLE peptides and corresponding receptor kinases, and the downstream signal transduction has been challenging due to their genome-wide redundancies and rapid evolution., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)