The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.     

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.     

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.     

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.     

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.     

On the constancy of cell division rate in the root meristem

This review examines under what circumstances the rate of cell division among cells of the root meristem is known to vary. First, methods are compared that have been used to quantify cell division rate. These can be grouped as being either cytological, in which the rate of accumulation of cells in a particular phase of the cell cycle is determined based on some form of cytological labeling, or kinematic, in which the rate of cell accumulation is determined from the net movement of cells. Then, evidence is reviewed as to whether cell division rates vary between different tissues or cell types, between different positions in the root, or finally between different environments. The evidence is consistent with cells dividing at a constant rate, and well documented examples where cell division rate changes substantially are rare. The constancy of cell division rate contrasts with the number of dividing cells, which varies extensively, and implies that a major point for cell cycle control is governing the exit from the proliferative state at the basal boundary of the meristem.     

A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem

Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor-like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor-like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor-like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage.     

In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis

The aerial parts of the plant are generated by groups of rapidly dividing cells called shoot apical meristems. To analyze cell behavior in these structures, we developed a technique to visualize living shoot apical meristems using the confocal microscope. This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerizing microtubules) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems. Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation were inhibited, suggesting an important role for DNA synthesis in growth and patterning.     

The effect of abscisic acid on cell growth, cell division and DNA synthesis in the maize root meristem

Cis -abscisic acid (ABA), when applied to maize ( Zea mays L. cv. LG 11) roots, decreases the rates of cell growth and cell division in the meristem. It also decreases the rate at which nuclei become labelled with [³H]-thymidine and enter mitosis. Removing the root cap accelerates the entry of nuclei into the DNA synthetic phase of the mitotic cycle and enhances the rate of cell proliferation in the quescent centre. ABA diminishes these effects, but does not suppress them. Thus, ABA cannot wholely substitute for the presence of a cap. One of the primary effects of applied ABA is to retard cell enlargement which may in turn affect the rate of cell division; natural endogenous ABA may act similarly. ABA might in this way assist in maintaining the quiescent centre in intact roots, but cannot be the sole agent involved.     

Thiol-containing molecules interact with the myeloperoxidase/H2O2/chloride system to inhibit LDL oxidation

Oxidized low-density lipoproteins (LDL) accumulate in the vascular wall and promote a local inflammatory process contributing to the progression of atheromatous plaque. The key role of myeloperoxidase (MPO) in this process has been documented and the enzyme has been involved in the oxidative modification of apolipoprotein B-100 in the intima and at the surface of endothelial cells. As the inhibition of this last phenomenon could be of relevance in pharmacological interventions, thiol-containing molecules such as glutathione, captopril, and N-acetylcysteine (NAC) and its lysinate salt (NAL) were tested in this system and their properties were compared with those of flufenamic acid (control). This last compound already demonstrated an inhibition of the production of HOCl by MPO and a more intense inhibition of MPO activity than glutathione, NAC, NAL, and captopril. However, NAC and NAL inhibited the oxidative modification of LDL more intensively than captopril and glutathione whereas flufenamic acid had no comparable inhibiting effect. This could be related to the presence of LDL close to the catalytic site of the enzyme. NAC and NAL therefore appeared as the most efficient inhibitors probably as a consequence of their relatively small size. The relevance of such effects has to be documented by in vivo studies.     

ATM/ATR-dependent Tyr15 phosphorylation of cyclin-dependent kinases in response to hydroxyurea in Vicia faba root meristem cells

DNA damage or stalled replication forks activate cell cycle checkpoints. However, the regulation of metabolic pathways that are responsible for maintenance of genome integrity in plants is still largely unknown. Present research on Vicia faba root meristem cells indicates that inhibitory phosphorylation of cyclin-dependent kinases (Cdks) at Tyr15 plays a prominent role during blockage of cell cycle in response to genotoxic stress. Phosphorylation of P-loop in Cdks takes place in ATM/ATR-dependent manner. Although, Tyr15 phosphorylation upon hydroxyurea (HU) treatment was found in most cells classified to G1 and S phase, interestingly, the number of phoshpo-Tyr15-positive cells decreases in G2 phase. Presented data confirm much similarity in regulation of Cdks functions under genotoxic stress between plants and animals; however, they may also substantiate evolutionarily developed differences especially in regulation of G2/M transition between these two kingdoms.     

ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem

It is well known that abscisic acid (ABA) can halt meristems for long periods without loss of meristem function, and can also promote root growth at low concentrations, but the mechanisms underlying such regulation are largely unknown. Here we show that ABA promotes stem cell maintenance in Arabidopsis root meristems by both promoting the quiescence of the quiescent centre (QC) and suppressing the differentiation of stem cells and their daughters. We demonstrate that these two mechanisms of regulation by ABA involve distinct pathways, and identify components in each pathway. Our findings demonstrate a cellular mechanism for a positive role for ABA in promoting root meristem maintenance and root growth in Arabidopsis.     

ROP GTPases act with the receptor-like protein PAN1 to polarize asymmetric cell division in maize

Plant Rho family GTPases (ROPs) have been investigated primarily for their functions in polarized cell growth. We previously showed that the maize (Zea mays) Leu-rich repeat receptor-like protein PANGLOSS1 (PAN1) promotes the polarization of asymmetric subsidiary mother cell (SMC) divisions during stomatal development. Here, we show that maize Type I ROPs 2 and 9 function together with PAN1 in this process. Partial loss of ROP2/9 function causes a weak SMC division polarity phenotype and strongly enhances this phenotype in pan1 mutants. Like PAN1, ROPs accumulate in an asymmetric manner in SMCs. Overexpression of yellow fluorescent protein-ROP2 is associated with its delocalization in SMCs and with aberrantly oriented SMC divisions. Polarized localization of ROPs depends on PAN1, but PAN1 localization is insensitive to depletion and depolarization of ROP. Membrane-associated Type I ROPs display increased nonionic detergent solubility in pan1 mutants, suggesting a role for PAN1 in membrane partitioning of ROPs. Finally, endogenous PAN1 and ROP proteins are physically associated with each other in maize tissue extracts, as demonstrated by reciprocal coimmunoprecipitation experiments. This study demonstrates that ROPs play a key role in polarization of plant cell division and cell growth and reveals a role for a receptor-like protein in spatial localization of ROPs.