Mis-expression of the CLV3/ESR-like gene CLE19 in Arabidopsis leads to a consumption of root meristem

Mild heat shock treatment (32 degrees C) of isolated Brassica napus microspores triggers a developmental switch from pollen maturation to embryo formation. This in vitro system was used to identify genes expressed in globular to heart-shape transition embryos. One of the genes isolated encodes a putative extra-cellular protein that exhibits high sequence similarity with the in silico identified CLV3/ESR-related 19 polypeptide from Arabidopsis (AtCLE19) and was therefore named BnCLE19. BnCLE19 is expressed in the primordia of cotyledons, sepals and cauline leaves, and in some pericycle cells in the root maturation zone. Mis-expression of BnCLE19 or AtCLE19 in Arabidopsis under the control of the CaMV 35S promoter resulted in a dramatic consumption of the root meristem, the formations of pin-shaped pistils and vascular islands. These results imply a role of CLE19 in promoting cell differentiation or inhibiting cell division.     

CLE14/CLE20 peptides may interact with CLAVATA2/CORYNE receptor-like kinases to irreversibly inhibit cell division in the root meristem of Arabidopsis

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.     

Root-specific CLE19 overexpression and the sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance

In the Arabidopsis shoot apical meristem, an organizing center signals in a non-cell-autonomous manner to specify the overlying stem cells. Stem cells express the small, secreted protein CLAVATA3 (CLV3; ) that activates the CLV1-CLV2 receptor complex, which negatively controls the size of the organizing center. Consistently, CLV3 overexpression restricts shoot meristem size. The root meristem also contains a stem cell organizer, and here we show that localized overexpression in roots of CLE19, encoding a CLV3 homolog, restricts the size of the root meristem. This suggests that CLE19 acts by overactivating an endogenous CLV-like pathway involved in root meristem maintenance. Surprisingly, CLE19 restricts meristem size without directly interfering with organizer and stem cell specification. We isolated mutations in two loci, SOL1 and SOL2, which suppress the CLE19 overexpression phenotype. sol2 plants display floral phenotypes reminiscent of clv weak alleles; these phenotypes suggest that components of a CLV pathway are shared in roots and shoots. SOL1 encodes a putative Zn(2+)-carboxypeptidase, which may be involved in ligand processing.     

Gene NANA regulates cell proliferation in Arabidopsis thaliana shoot apical meristem without interaction with CLV1, CLV2, CLV3

A constancy of stem cell pool in shoot apical meristem of Arabidopsis thaliana is provided by a genetic regulation system with negative feedback loop based on the interaction of the gene WUS, which maintains indeterminate state of cells, with CLV genes, which restrict the level of WUS expression and stem cell pool size. clv mutations lead to an increase in the pool of stem cells in the apical and floral meristems and wus mutation leads to the opposite effect. Mutation na (nana), like wus mutation, causes premature termination of shoot apical meristem function, although it does not affect the activity of the flower meristem. To elucidate the role of NA in the control of shoot apical meristem functioning, the interaction of NA with CLV genes were investigated. Additive phenotype of double mutants na clv1, na clv2-1, and na clv3-2 indicates that the NA gene makes an independent contribution to the functioning of the shoot apical meristem. It is assumed that the NA gene controls apical meristem cell proliferation during the transition to the reproductive phase of plant development, acting much later and independently of the genes WUS-CLV.     

CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex

The phloem, located within the vascular system, is critical for delivery of nutrients and signaling molecules throughout the plant body. Although the morphological process and several factors regulating phloem differentiation have been reported, the molecular mechanism underlying its initiation remains largely unknown. Here, we report that the small peptide‐coding gene, CLAVATA 3 (CLV3)/EMBEYO SURROUNDING REGION 25 (CLE25), the expression of which begins in provascular initial cells of 64‐cell‐staged embryos, and continues in sieve element‐procambium stem cells and phloem lineage cells, during post‐embryonic root development, facilitates phloem initiation in Arabidopsis. Knockout of CLE25 led to delayed protophloem formation, and in situ expression of an antagonistic CLE25_G6T peptide compromised the fate‐determining periclinal division of the sieve element precursor cell and the continuity of the phloem in roots. In stems of CLE25_G6T plants the phloem formation was also compromised, and procambial cells were over‐accumulated. Genetic and biochemical analyses indicated that a complex, consisting of the CLE‐RESISTANT RECEPTOR KINASE (CLERK) leucine‐rich repeat (LRR) receptor kinase and the CLV2 LRR receptor‐like protein, is involved in perceiving the CLE25 peptide. Similar to CLE25, CLERK was also expressed during early embryogenesis. Taken together, our findings suggest that CLE25 regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex.     

CLV3 is localized to the extracellular space,where it activates the Arabidopsis CLAVATA stem cell signaling pathway

Plant growth and development depends on the activity of a continuously replenished pool of stem cells within the shoot apical meristem to supply cells for organogenesis. In Arabidopsis, the stem cell-specific protein CLAVATA3 (CLV3) acts cell nonautonomously to restrict the size of the stem cell population, but the hypothesis that CLV3 acts as an extracellular signaling molecule has not been tested. We used genetic and immunological assays to show that CLV3 localizes to the apoplast and that export to the extracellular space is required for its function in activating the CLV1/CLV2 receptor complex. Apoplastic localization allows CLV3 to signal from the stem cell population to the organizing center in the underlying cells.     

Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-related (CLE) peptides in Arabidopsis thaliana and Oryza sativa

Using 26 chemically synthetic CLAVATA3/ESR (CLE) peptides, which correspond to the predicted products of the 31 Arabidopsis CLE genes, we investigated the CLE peptide function in Arabidopsis and rice. Treatment with some CLE peptides inhibited root elongation in rice as well as in Arabidopsis. It also reduced the size of the shoot apical meristem in Arabidopsis but not in rice. Database searches revealed 47 putative CLE genes in the rice genome and multiple CLE domains in some CLE genes, indicating diverse CLE function in these plants.     

Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis

As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.     

The Arabidopsis CLV3-like (CLE) genes are expressed in diverse tissues and encode secreted proteins

Members of the receptor-like kinase gene family play crucial regulatory roles in many aspects of plant development, but the ligands to which they bind are largely unknown. In Arabidopsis, the receptor kinase CLAVATA1 (CLV1) binds to the small secreted polypeptide CLV3, and three proteins act as key elements of a signal transduction pathway that regulates shoot apical meristem maintenance. To better understand the signal transduction mechanisms involving small polypeptides, we are studying 25 Arabidopsis CLV3/ESR (CLE) proteins that share a conserved C-terminal domain with CLV3 and three maize ESR proteins. Members of the CLE gene family were identified in database searches and only a few are known to be expressed. We have identified an additional member of the CLE gene family in Arabidopsis, which is more similar in gene structure to CLV3 than the other CLE genes. Phylogenetic analysis reveals that few of the putative CLE gene products are closely related, suggesting there may be little functional overlap between them. We show that 24 of the 25 Arabidopsis CLE genes are transcribed in one or more tissues during development, indicating that they do encode functional products. Many are widely expressed, but others are restricted to one or a few tissue types. We have also determined the sub-cellular localization of several CLE proteins, and find that they are exported to the plasma membrane or extracellular space. Our results suggest that the Arabidopsis CLE proteins, like CLV3, may function as secreted signaling molecules that act in diverse pathways during growth and development.     

The CLAVATA1-related BAM1, BAM2 and BAM3 receptor kinase-like proteins are required for meristem function in Arabidopsis

Organ formation at shoot and flower meristems in plants requires the maintenance of a population of centrally located stem cells and the differentiation of peripherally located daughter cells. The CLAVATA (CLV) gene products in Arabidopsis, including the CLV1 receptor-kinase, regulate this process by promoting the differentiation of stem cells on the meristem flanks. Here, we have analyzed the developmental roles of the CLV1-related BAM1 (derived from barely any meristem 1), BAM2 and BAM3 receptor-like kinases. Loss-of-function alleles of these receptors lead to phenotypes consistent with the loss of stem cells at the shoot and flower meristem, suggesting that their developmental role is opposite to that of CLV1. These closely related receptors are further distinguished from CLV1, whose expression and function is highly specific, by having broad expression patterns and multiple developmental roles. These include a requirement for BAM1, BAM2 and BAM3 in the development of high-ordered vascular strands within the leaf and a correlated control of leaf shape, size and symmetry. In addition, BAM1, BAM2 and BAM3 are required for male gametophyte development, as well as ovule specification and function. Significantly, the differing roles of CLV1 and BAM receptors in meristem and organ development are largely driven by differences in expression patterns.     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CKV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the "reaction-diffusion" type formalizing the role proteins CLV1, CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CLV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the “reaction-diffusion” type formalizing the role proteins CLV1/CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

The role of CLV1, CLV2 and HPAT homologues in the nitrogen-regulation of root development

Plants use a variety of signals to control root development, including in modifying root development in response to nutrient stress. For example, in response to nitrogen (N) stress, plants dramatically modulate root development, including the formation of N-fixing nodules in legumes. Recently, specific CLE peptides and/or receptors important for their perception, including CLV1 and CLV2, have been found to play roles in root development, including in response to N supply. In the legume Medicago truncatula, this response also appears to be influenced by RDN1, a member of the hydroxyproline-O-arabinosyltransferase (HPAT) family which can modify specific CLE peptides. However, it is not known if this signalling pathway plays a central role in root development across species, and in particular root responses to N. In this study, we systematically examined the role of the CLV signalling pathway genes in root development of the legume pea (Pisum sativum) and non-legume tomato (Solanum lycopersicum) using a mutant-based approach. This included a detailed examination of root development in response to N in tomato mutants disrupted in CLV1- or CLV2-like genes or HPAT family member FIN. We found no evidence for a role of these genes in pea seedling root development. Furthermore, the CLV1-like FAB gene did not influence tomato root development, including the root response to N supply. In contrast, both CLV2 and the HPAT gene FIN appear to positively influence root size in tomato but do not mediate root responses to N. These results suggest the function of these genes may vary somewhat in different species, including the N regulation of root architecture.     

Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co‐immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2–CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two‐parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.     

AHK5 histidine kinase regulates root elongation through an ETR1-dependent abscisic acid and ethylene signaling pathway in Arabidopsis thaliana

The Arabidopsis thaliana genome encodes a small family of histidine (His) protein kinases, some of which have redundant functions as ethylene receptors, whereas others serve as cytokinin receptors. The most poorly characterized of these is authentic histidine kinase 5 (AHK5; also known as cytokinin-independent 2, CKI2). Here we characterize three independent ahk5 mutants, and show that they have a common phenotype. Our results suggest that AHK5 His-kinase acts as a negative regulator in the signaling pathway in which ethylene and ABA inhibit the root elongation through ETR1 (an ethylene receptor).