Nitric oxide production by the differentiating xylem of Zinnia elegans

Nitric oxide (NO) is currently regarded as a signal molecule involved in plant cell differentiation and programmed cell death. Here, we investigated NO production in the differentiating xylem of Zinnia elegans by confocal laser scanning microscopy to answer the question of whether NO is produced during xylem differentiation. Results showed that NO production was mainly located in both phloem and xylem regardless of the cell differentiation status. However, there was evidence for a spatial NO gradient inversely related to the degree of xylem differentiation and a protoplastic NO burst was associated with the single cell layer of pro-differentiating thin-walled xylem cells. Confirmation of these results was obtained using trans-differentiating Z. elegans mesophyll cells. In this system, the scavenging of NO by means of 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO) inhibits tracheary element differentiation but increases cell viability. These results suggest that plant cells, which are just predetermined to irreversibly trans-differentiate in xylem elements, show a burst in NO production, this burst being sustained as long as secondary cell wall synthesis and cell autolysis are in progress.     

Effects of cell tension on the small GTPase Rac

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.     

The small GTPase Rab2 functions in the removal of apoptotic cells in Caenorhabditis elegans

We identify here a novel class of loss-of-function alleles of uncoordinated locomotion(unc)-108, which encodes the Caenorhabditis elegans homologue of the mammalian small guanosine triphosphatase Rab2. Like the previously isolated dominant-negative mutants, unc-108 loss-of-function mutant animals are defective in locomotion. In addition, they display unique defects in the removal of apoptotic cells, revealing a previously uncharacterized function for Rab2. unc-108 acts in neurons and engulfing cells to control locomotion and cell corpse removal, respectively, indicating that unc-108 has distinct functions in different cell types. Using time-lapse microscopy, we find that unc-108 promotes the degradation of engulfed cell corpses. It is required for the efficient recruitment and fusion of lysosomes to phagosomes and the acidification of the phagosomal lumen. In engulfing cells, UNC-108 is enriched on the surface of phagosomes. We propose that UNC-108 acts on phagosomal surfaces to promote phagosome maturation and suggest that mammalian Rab2 may have a similar function in the degradation of apoptotic cells.     

The small GTPase Rac1 is involved in the maintenance of stemness and malignancies in glioma stem-like cells

A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance, and malignant progression; however, the molecular mechanisms underlying the maintenance of cancer stem-like cell properties have remained unclear. Here, we show that the Rho family GTPase Rac1 is involved in the glioma stem-like cell (GSLC) maintenance and tumorigenicity in human glioma. The Rac1-Pak signaling was markedly activated in GSLCs. Knockdown of Rac1 caused reduction of expression of GSLC markers, self-renewal-related proteins and neurosphere formation. Moreover, down-regulation of Rac1 suppressed the migration, invasion, and malignant transformation in GSLCs. Furthermore, inhibition of Rac1 enhanced radiation sensitivity of GSLCs. These results indicate that the small GTPase Rac1 is involved in the maintenance of stemness and malignancies in GSLCs.     

Role of the small GTPase Rac in p22phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22(phox), which provides both stabilization and a docking site for organizer proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox) constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67(phox). Since the enhancements are not observed with a mutant p67(phox) defective in binding to Rac, this GTPase appear to directly recruit p67(phox) to the membrane.     

Activation of the small GTPase Rac1 by cGMP-dependent protein kinase

Cyclic-GMP-dependent protein kinase (PKG) is widely appreciated as having diverse roles in a variety of cell types. Many reports have indicated that PKG might regulate cell function by activating members of the mitogen-activated protein kinase (MAPK) family of signaling proteins. In this study, stimulation of HEK-293 cells with nitric oxide (NO) was found to induce a rapid accumulation of phosphorylated p38 MAPK. The involvement of PKG in this process was confirmed by cotransfection of a dominant negative PKG construct (G1alphaR-GFP), which was able to block cGMP-induced p38 MAPK activation. Transfection of cells to express dominant negative Rac1(T17N) was also able to dose-dependently block cGMP-stimulated activation of p38 MAPK, thus indicating the importance of this pathway downstream of PKG. GST-PDB affinity-precipitation experiments revealed that stimulation of HEK293 cells with either nitric oxide or 8-Br-cGMP resulted in a rapid and transient activation of Rac1 with similar kinetics to p38 MAPK phosphorylation. Moreover, using in vitro kinase assays it was found that cGMP also stimulated the activity of the Rac1 effector Pak1. The activation of both Rac1 and Pak1 by 8-Br-cGMP was completely abolished by transfection of the cells with G1alphaR-GFP. Expression of the Rac1(T17N) mutant inhibited PKG-dependent activation of PAK1 indicating that Rac1 functions upstream of PAK1 in this pathway. Immunofluorescence experiments demonstrated clear colocalization of PKG and Rac1 in membrane ruffles and dynamic membrane regions supporting a functional interaction. However, in vitro kinase assays demonstrated that Rac1 is not a substrate for PKG suggesting an indirect activation mechanism. Taken together these data demonstrate a novel PKG-dependent pathway by which the Rac1/Pak1 pathway is activated. Furthermore, we demonstrate that this pathway is central to the activation of p38 MAPK by PKG in these cells.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells.

BACKGROUND: Tracheary element (TE) differentiation in single cells in culture isolated from Zinnia elegans leaves involves programmed cell death (PCD) co-ordinated with key morphological developments. We have used flow cytometry to analyze physiological and nuclear changes in the differentiating cells. Flow cytometry allows the identification of subpopulations, thereby removing the obscuring effect of population heterogeneity that occurs with the use of other techniques. METHODS: Cell viability, plasma membrane integrity, oxidative activity, intracellular calcium and pH, cell wall thickening, the possible role of microtubule rearrangement, chromatin condensation, and DNA breakdown were followed by flow cytometry from the first stages of TE induction. RESULTS: TE differentiation could be enhanced and made more synchronous by a centrifugation step at 72 h after cell isolation. Size and shape changes were the first changes identified in differentiating cells, and these properties could be used to isolate differentiating populations by back-gating. Chromatin condensation and nDNA breakdown followed patterns characteristic of programmed cell death. CONCLUSIONS: We have used flow cytometry to characterize the morphological and physiological changes that occur during TE differentiation, and our findings indicate that this process is a form of autophagic PCD in which microtubule rearrangement appears to play a role.     

Connections between integrins and Rac GTPase pathways control gonad formation and function in C. elegans

The integrins are a family of alphabeta heterodimeric transmembrane receptors that link extracellular matrix (ECM) proteins to the cytoskeleton and orchestrate cell behaviors. It's been suggested that integrins interact with Rho family small GTPases, such as Rho and Rac. We took advantage of a C. elegans nematode line expressing HA-betatail, a beta integrin transgene inhibiting the functions of endogenous integrins, to determine the combined effects of reducing PAT-3 beta integrin and Rac pathway activities. Double mutants of HA-betatail and unc-73, a guanine nucleotide exchange factor GEF for MIG-2/Rac, had body wall and vulval muscle abnormalities. On the other hand, HA-betatail combined with mutant CED-5, another Rac interacting protein, showed ovulation defects and sterility. RNA-mediated interference (RNAi) of pat-3 on Rac mutant backgrounds also affected gonad structure and function. These results show a functional link between integrins and Rac signaling in muscles and gonads. Furthermore, data showing distinct phenotypes of HA-betatail with unc-73 versus ced-5 suggest some tissue-specificity in the usage of Rac signaling pathways.     

Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells

Hypoxia-induced angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Rho family small GTPases are involved in this process, and Racl, a prominent member of the Rho family, may be critical in regulating hypoxia-induced gene activation of several angiogenesis factors and tumor suppressors. To fur-ther define Racl function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock down gene expression of Racl in gastric cancer cell line AGS that expresses a high level of Racl.Both the mRNA and protein levels of Racl in the AGS cells were decreased dramatically after transfection with a Racl-specific siRNA vector. When the conditioned medium derived from the Racl downregulated AGS cells was applied to the human endothelial cells. it could significantly inhibit the cell proliferation. Further study proved that, VEGF and HIF-la, two angiogenesis promoting factors, were found to be downregulated whereas p53 and VHL, which are tumor suppressors and angiogenesis inhibitors. were upregulated in the Racl siRNA transfected cells. Our results suggest that Racl may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting the Racl GTPase.     

Zinnia elegans uses the same peroxidase isoenzyme complement for cell wall lignification in both single-cell tracheary elements and xylem vessels

The nature of the peroxidase isoenzyme complement responsible for cell wall lignification in both Zinnia elegans seedlings and Z. elegans tracheary single-cell cultures have been studied. Results showed that both hypocotyls and stems from lignifying Z. elegans seedlings express a cell wall-located basic peroxidase of pI approximately 10.2, which was purified to homogeneity. Molecular mass determination under non-denaturing conditions showed an M(r) of about 43 000, similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496-501 and 632-635 (alpha and beta absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group being ferric protoporphyrin IX. The N-terminal amino acid sequence of this Z. elegans basic peroxidase was KVAVSPLS (peptide motif in bold), which shows strong homologies with the N-amino acid terminus of other strongly basic plant peroxidases. Isoenzyme and western blot analyses showed that this peroxidase isoenzyme is also expressed in trans-differentiating Z. elegans tracheary single-cell cultures. The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. From these results, it may be concluded that Z. elegans uses a single programme, i.e. an identical peroxidase isoenzyme complement, for lignification of the xylem, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels).     

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ～100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.     

Chitosan and a fungal elicitor inhibit tracheary element differentiation and promote accumulation of stress lignin-like substance in Zinnia elegans xylogenic culture

We investigated the effect of elicitors on xylem differentiation and lignification using a Zinnia elegans xylogenic culture system. Water-soluble chitosan and a fungal elicitor derived from Botrytis cinerea were used as elicitors. Elicitor addition at the start of culturing inhibited tracheary element (TE) differentiation in a concentration-dependent manner, and 30 μg mL^?1 of chitosan or 16.7 μg mL^?1 of the fungal elicitor strikingly inhibited TE differentiation and lignification. Addition of chitosan (at 50 μg mL^?1) or the fungal elicitor (at 16.7 μg mL^?1) during the culturing period also inhibited TE differentiation without inhibiting cell division, except for immature TEs undergoing secondary wall thickening. Elicitor addition after immature TE appearance also caused the accumulation of an extracellular lignin-like substance. It appears that elicitor addition at the start of culturing inhibits the process by which dedifferentiated cells differentiate into xylem cell precursors. Elicitor addition during culturing also appears to inhibit the transition from xylem cell precursors to immature TEs, and induces xylem cell precursors or xylem parenchyma cells to produce an extracellular stress lignin-like substance.     

ATPase in mature and differentiating phloem and xylem

A cytochemical study using a lead precipitation technique has been made of the distribution of adenosine triphosphatase (ATPase) in mature and differentiating phloem and xylem cells of Nicotiana tabacum and Pisum sativum. The sites of ATPase localization in tobacco phloem were the plasma membrane, endoplasmic reticulum, mitochondria, dictyosomes, plasmodesmata, and the dispersed P proteins of mature sieve elements. In pea phloem sieve elements ATPase was localized in the endoplasmic reticulum, but was not associated with the P proteins or plasma membranes at any stage of their differentiation. In pea transfer cells ATPase activity was associated with the endoplasmic reticulum at all stages of their differentiation and with the plasma membrane of transfer cells that had formed wall ingrowths. In xylem cells of both tobacco and pea the patterns of ATPase activity was similar. At early stages of differentiation ATPase activity was associated with the plasma membrane and the endoplasmic reticulum. At intermediate stages of differentiation ATPase activity continued to be associated with the endoplasmic reticulum, but was no longer associated with the plasma membrane. At later stages of xylem element differentiation ATPase activity was associated with disintegrating organelles and with the hydrolyzing cell walls.     

Differences in response to colchicine by differentiating xylem cells in roots ofPisum

Summary A 3 hour exposure of pea roots to 0.025 per cent colchicine causes the cessation of root growth and ac-tumour to form. In roots allowed to regenerate it is found that in the region of thec-tumour, metaxylem tissue fails to differentiate completely, but protoxylem is unaffected. Development of secondary xylem tissue is also inhibited in the tumour region.     

Alternative splicing of Rac1 generates Rac1b, a self-activating GTPase

Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP- and the GppNHp-bound forms, determined at a resolution of 1.75 A, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPase-binding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase.     

Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac

Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.     

Involvement of phosphoinositide turnover in tracheary element differentiation in Zinnia elegans L. cells

Mesophyll cells of Zinnia elegans L., cultured in the presence of phytohormones, will transdifferentiate and undergo programmed cell death to become tracheary elements, thick-walled cells of the xylem. This system is a model system for study of plant cell development and differentiation. We report that a high concentration of extracellular Ca(2+) is necessary during the first 6 h of culturing for tracheary elements to form. Extracellular Ca(2+) is still required at later times, but at a much lower concentration. When cells transdifferentiate in adequate Ca(2+), microsomal phospholipase C activity increases and levels of inositol 1,4,5-trisphosphate rise at about hour 4 of culturing. The production of inositol 1,4,5-trisphosphate appears to be important for tracheary element formation, since inhibitors of phospholipase C inhibit both inositol 1,4,5-trisphosphate production and tracheary element formation. Pertussis toxin, an inhibitor of GTP-binding proteins, inhibits transdifferentiation and eliminates inositol 1,4,5-trisphosphate production. Tracheary element formation was not completely abolished by inhibitors that eliminated inositol 1,4,5-trisphosphate production, suggesting the involvement of other pathways in regulating transdifferentiation.