The role of nodal and internodal responses in gravitropism and autotropism in Galium aparine L

Abstract This time course and location of gravitropically induced curvatures in stems of goosegrass (Galium aparine L.), a member of the Rubiaceae, have been investigated. In the early stages of the response (0–5 h), curvature develops throughout the growing region, and is followed by an autotropic straightening which affects the internodes only, leading to the production of essentially straight internodes some 15 h after the onset of gravistimulation. Curvatures developing in the nodal regions, however, continue to increase over this period, and are not subject to reversal by autotropism. The nodal curvatures are not entirely dependent on the presence of any other part of the plant, since marked curvatures can be induced in isolated nodal segments. This pattern of response leads ultimately to correction of the growth direction of the plant by means of curvature responses confined exclusively to the nodes, despite the initial participation of both nodes and internodes in the gravitropic reaction.     

Endogenous ethylene does not regulate opening of unstressed Iris flowers but strongly inhibits it in water-stressed flowers

The floral buds of Iris flowers (Iris x hollandica) are enclosed by two sheath leaves. Flower opening depends on lifting the flower up to a position whereby the tepals can move laterally. This upward movement is carried out by elongation of the subtending pedicel and ovary. In the pedicels and ovaries of unstressed control flowers, the concentration of ACC (1-aminocyclopropane-1-carboxylic acid) and the rate of ethylene production increased during d 0-1 of flower opening, and then decreased. Exposure to ≥200nLL(-1) ethylene for 24h at 20°C inhibited elongation of the pedicel+ovary, and inhibited flower opening. However, pulsing of unstressed flowers with solutions containing inhibitors of ethylene synthesis (AOA, AVG), or an inhibitor of ethylene action (STS), did not affect pedicel+ovary elongation or flower opening. When the flowers were dehydrated for 2 d at 20°C and 60% RH, they did not open when subsequently placed in water, and showed inhibited elongation in the pedicel+ovary. This dehydration treatment resulted in elevated pedicel+ovary ACC levels and in increased ethylene production. Treatment with STS prevented the increase in ACC levels and ethylene production, overcame the effect of dehydration on elongation of the pedicel+ovary, and resulted in full flower opening. It is concluded that flower opening in unstressed Iris flowers is not regulated by endogenous ethylene. An increase in endogenous ethylene above normal levels during stress, by contrast, strongly inhibited flower opening, due to its inhibitory effect on elongation of the pedicel+ovary.     

Seasonal dynamics of carbohydrate and nitrogenous components in the roots of perennial weeds

Abstract Chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale L.) are persistent weeds, the aerial portions of which do not survive in winter. However, subterranean tissues remain viable and facilitate the rapid resumption of growth in early spring. The source of nutrients for growth prior to the establishment of foliage is the roots. Carbohydrate and N reserves are accrued during late summer and autumn, respectively. Hydrolysis of fructans during late autumn occurs coincidentally with increments in sucrose, the latter providing a readily accessible C pool. Nitrate, free amino acids and soluble protein all play substantial roles in nitrogen storage. Asparagine is the predominant amino acid in the free pool during winter, followed by glutamine, ornithine, serine, aspartic acid and glutamic acid. Storage reserves remain at peak levels throughout winter and deeline prior to the resumption of growth. The patterns observed here provide evidence that N is an important currency of storage metabolism and, thus, a framework has been provided for the examination of regulation of N storage in perennial weeds.     

The presence of PEG-lipids in liposomes does not reduce melittin binding but decreases melittin-induced leakage

Poly(ethyleneglycol) (PEG), anchored at the surface of liposomes via the conjugation to a lipid, is commonly used for increasing the liposome stability in the blood stream. In order to gain a better understanding of the protective properties of interfacial polymers, we have studied the binding of melittin to PEG-lipid-containing membranes as well as the melittin-induced efflux of a fluorescent marker from liposomes containing PEG-lipids. We examined the effect of the polymer size by using PEG with molecular weights of 2000 and 5000. In addition, we studied the role of the anchoring lipid by comparing PEG conjugated to phosphatidylethanolamine (PE) which results in a negatively charged PEG-PE, with PEG conjugated to ceramide (Cer) which provides the neutral PEG-Cer. Our results show that interfacial PEG does not prevent melittin adsorption onto the interface. In fact, PEG-PE promotes melittin binding, most likely because of attractive electrostatic interactions with the negative interfacial charge density of the PEG-PE-containing liposomes. However, PEG-lipids limit the lytic potential of melittin. The phenomenon is proposed to be associated with the change in the polymorphic tendencies of the liposome bilayers. The present findings reveal that the protective effect associated with interfacial hydrophilic polymers is not universal. Molecules like melittin can sense surface charges borne by PEG-lipids, and the influence of PEG-lipids on liposomal properties such as the polymorphic propensities may be involved in the so-called protective effect.     

The role of methionine recycling for ethylene synthesis in Arabidopsis

The methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from S-adenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 mum sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfur-limiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.     

A method to study slug predation on seedlings in the field

Dandelion is a common Asteraceae species that populates disturbed sites and gaps within swards where it becomes an important competitor of grasses. The natural control against dandelion includes seedling predation, with slugs, particularly Arion lusitanicus, being the most important in the Czech Republic. However, the study of slug seedling consumption is difficult because naturally established seedlings are not always available. Therefore, we developed a method of exposing laboratory‐grown seedlings as bait for slug predation. Dandelion seeds were sown in plastic cups containing a bottom layer of moist substrate. The emerged seedlings were thinned to 20 and displayed in an open area. During 2008–2010, the seedling baits were placed at 15 sites at 1 month intervals throughout the dandelion vegetative season, in parallel with plasticine baits that monitored slug feeding activity. For each 1 month interval, seedling survival was observed for a period of 8 days, and the estimated time to death was calculated; the percentage of surviving seedlings was then recorded. This method of seedling presentation demonstrated that local and temporal variation in seedling survival is correlated with slug feeding activity. The advantage of this technique is that the seedlings in baits may be presented at any place and time, as required by the experimental design; however, we found that the estimated time to seedling death was shorter for the exposed baits than for the naturally established seedlings. The method is suitable for the study of plant species other than dandelion, and also for aims other than the study of spatiotemporal trends in seedling consumption by slugs.     

Acid sphingomyelinase is induced by butyrate but does not initiate the anticancer effect of butyrate in HT29 and HepG2 cells

Butyric acid and sphingomyelin (SM) affect colonic tumorigenesis. We examined the potential link between butyrate stimulation and SM metabolism in colonic and hepatic cancer cell lines. After incubating HT29 and HepG2 cells with butyrate and other short-chain fatty acids, we found that butyrate increased acid but not neutral or alkaline sphingomyelinase (SMase) activity by 10- to 20-fold. The effects occurred after 16 h of incubation and were associated with reduced SM and phosphatidylcholine contents and increased ceramide levels. Northern blotting showed increased acid SMase mRNA levels in these cells after butyrate stimulation. Propionate was less potent, and acetate had no effect. No similar changes of acid phosphatase could be identified. At concentrations that increased acid SMase expression, butyrate inhibited cell proliferation, activated caspase 3, and induced apoptosis. However, the antiproliferative and apoptotic effects of butyrate preceded the changes of acid SMase and were not affected by knocking down acid SMase expression by small, interfering RNA. In addition, butyrate-induced acid SMase expression was not affected by blocking the caspase pathway. In conclusion, butyrate regulates SM metabolism by stimulating acid SMase expression in colon and liver cancer cells, but the increased acid SMase is not a critical mechanism for initiating the anticancer effects of butyrate.     

A possible regulatory role for ethylene in the pollen-pistil interaction in a sporophytic self-incompatible system

The sporophytic type of self-incompatibility exhibited by Ipomoea cairica Sweet (Convolvulaceae) was partially overcome in vitro by treating the pollen and/or stigma with 10^–6 to 10^–1 M methionine, a precursor of ethylene. The implications of these observations are discussed in relation to other experiments involving use of the ethylene antagonist AgNO₃, individually and in combination with methionine and an optimum level of indole-3-acetic acid (10^–2 M). The results suggest a role for ethylene (which could also be IAA-induced) in regulating pollen germination and further tube growth in sporophytic self-incompatible systems. A hypothesis on the action of hormones in pollen germination and tube growth in a sporophytic self-incompatible (SSI) system is presented.     

A direct role for Fgf but not Wnt in otic placode induction

Induction of the otic placode, which gives rise to all tissues comprising the inner ear, is a fundamental aspect of vertebrate development. A number of studies indicate that fibroblast growth factor (Fgf), especially Fgf3, is necessary and sufficient for otic induction. However, an alternative model proposes that Fgf must cooperate with Wnt8 to induce otic differentiation. Using a genetic approach in zebrafish, we tested the roles of Fgf3, Fgf8 and Wnt8. We demonstrate that localized misexpression of either Fgf3 or Fgf8 is sufficient to induce ectopic otic placodes and vesicles, even in embryos lacking Wnt8. Wnt8 is expressed in the hindbrain around the time of otic induction, but loss of Wnt8 merely delays expression of preotic markers and otic vesicles form eventually. The delay in otic induction correlates closely with delayed expression of fgf3 and fgf8 in the hindbrain. Localized misexpression of Wnt8 is insufficient to induce ectopic otic tissue. By contrast, global misexpression of Wnt8 causes development of supernumerary placodes/vesicles, but this reflects posteriorization of the neural plate and consequent expansion of the hindbrain expression domains of Fgf3 and Fgf8. Embryos that misexpress Wnt8 globally but are depleted for Fgf3 and Fgf8 produce no otic tissue. Finally, cells in the preotic ectoderm express Fgf (but not Wnt) reporter genes. Thus, preotic cells respond directly to Fgf but not Wnt8. We propose that Wnt8 serves to regulate timely expression of Fgf3 and Fgf8 in the hindbrain, and that Fgf from the hindbrain then acts directly on preplacodal cells to induce otic differentiation.     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

A role for cyanide, derived from ethylene biosynthesis, in the development of stress symptoms

Cyanide is formed as a co-product of ethylene during the oxidation of 1-aminocyclo-propane-1-carboxylic acid (ACC) catalyzed by ACC oxidase. A toxic or regulatory function for cyanide in plant metabolism remains controversial. However, recent studies on the mode of action of auxin herbicides in sensitive plants suggest that the accumulation of tissue cyanide, derived ultimately from herbicide-stimulated ACC synthesis, is implicated in the induction of herbicide phytotoxicity. Furthermore, increases in cyanide levels have been observed during the formation of necrotic lesions in tobacco mosaic virus-infected tobacco leaves. It thus appears worthwhile to elucidate in more detail a possible role for cyanide in the induction of cell death under stress conditions which coincide with a strong stimulation of ethylene biosynthesis.     

Mild mitochondrial uncoupling does not affect mitochondrial biogenesis but downregulates pyruvate carboxylase in adipocytes: role for triglyceride content reduction

In adipocytes, mitochondrial uncoupling is known to trigger a triglyceride loss comparable with the one induced by TNFα, a proinflammatory cytokine. However, the impact of a mitochondrial uncoupling on the abundance/composition of mitochondria and its connection with triglyceride content in adipocytes is largely unknown. In this work, the effects of a mild mitochondrial uncoupling triggered by FCCP were investigated on the mitochondrial population of 3T3-L1 adipocytes by both quantitative and qualitative approaches. We found that mild mitochondrial uncoupling does not stimulate mitochondrial biogenesis in adipocytes but induces an adaptive cell response characterized by quantitative modifications of mitochondrial protein content. Superoxide anion radical level was increased in mitochondria of both TNFα- and FCCP-treated adipocytes, whereas mitochondrial DNA copy number was significantly higher only in TNFα-treated cells. Subproteomic analysis revealed that the abundance of pyruvate carboxylase was reduced significantly in mitochondria of TNFα- and FCCP-treated adipocytes. Functional study showed that overexpression of this major enzyme of lipid metabolism is able to prevent the triglyceride content reduction in adipocytes exposed to mitochondrial uncoupling or TNFα. These results suggest a new mechanism by which the effects of mitochondrial uncoupling might limit triglyceride accumulation in adipocytes.     

A primary role for microfilaments, but not microtubules, in hormone-induced cytoplasmic retraction

The distributions of microfilaments and microtubules were studied during transient hormone-induced changes in cell shape (retraction-respreading). Two cell types (fibroblasts and bone cells), differentially responsive to parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂), were analysed. The cytoplasm of fibroblasts retracted in response to PGE₂ but not PTH, whereas bone cells could respond to both PGE₂ and PTH. Time-lapse photomicrography indicated that the retraction began within minutes of hormone addition, while respreading occurred over longer times, up to 8 h. Affinity-purified actin and tubulin antibodies were used to follow the appearance of microtubules and microfilaments during both the retraction and the respreading phases. Microtubules appeared not to reorganize noticeably, although they were squeezed closer together in cellular pseudopods; no extensive loss or growth was detectable. Microfilaments did alter drastically their appearance and distributions. Soon after hormone addition when earliest detectable cytoplasmic retraction was evident, microfilament bundles appeared to break down. Remaining microfilament bundles consisted of relatively short, non-aligned fragments or aggregates. During respreading, microfilament bundles regrew and realigned throughout the cytoplasm. These data suggest a primary role for microfilaments, but probably not microtubules, in these cell shape changes.     

Intermediate frequency magnetic field at 23 kHz does not modify gene expression in human fetus‐derived astroglia cells

The increased use of induction heating (IH) cooktops in Japan and Europe has raised public concern on potential health effects of the magnetic fields generated by IH cooktops. In this study, we evaluated the effects of intermediate frequency (IF) magnetic fields generated by IH cooktops on gene expression profiles. Human fetus‐derived astroglia cells were exposed to magnetic fields at 23 kHz and 100 µT_rms for 2, 4, and 6 h and gene expression profiles in cells were assessed using cDNA microarray. There were no detectable effects of the IF magnetic fields at 23 kHz on the gene expression profile, whereas the heat treatment at 43 °C for 2 h, as a positive control, affected gene expression including inducing heat shock proteins. Principal component analysis and hierarchical analysis showed that the gene profiles of IF‐exposed groups were similar to the sham‐exposed group and were different than the heat treatment group. These results demonstrated that exposure of human fetus‐derived astroglia cells to an IF magnetic field at 23 kHz and 100 µT_rms for up to 6 h did not induce detectable changes in gene expression profile. Bioelectromagnetics 33:662–669, 2012. © 2012 Wiley Periodicals, Inc.     

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model

We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and “purse string”-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent “purse string”-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype. This work was supported by grants from the Faculty of Science at Toho University and from Fujirebio to M.S.     

Atorvastatin and losartan may upregulate renalase activity in hypertension but not coronary artery diseases: The role of gene polymorphism

The aim is to explore the treatment effect of coronary artery disease (CAD) and hypertension on plasma levels of renalase activity and also the possible association of renalase rs10887800 gene polymorphism with CAD and hypertension. A total of 286 patients who received coronary angiography were included in the study. Subjects were divided into four groups including (1) hypertensive with no CAD (H-Tens, n = 60); (2) CAD with hypertension (CAD + H-Tens, n = 71); (3) CAD with no hypertension (CAD, n = 61); and (4) nonhypertensive with no CAD as a control group (Con, n = 69). The plasma renalase activity was measured using the Amplex Red Monoamine Oxidase Assay Kit. Renalase rs10887800 single-nucleotide polymorphisms (SNPs) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Atorvastatin (P = 0.005), losartan (P < 0.001), and captopril (P = 0.001) were administered significantly more in case groups compared with the Con group. Significant higher and lower levels of renalase activity were observed in H-Tens and CAD patients compared with control subjects (P < 0.001 for both comparisons). Furthermore, no significant differences were obtained in the risk or protective effects of renalase rs10887800 SNP against hypertension and/or CAD in both recessive and dominant genetic models (P > 0.05). According to the findings of the present study, atorvastatin and losartan therapy assumes considerable significance in alleviating hypertension, but not CAD, by increasing the renalase activity. Furthermore, it was found that renalase rs10887800 is less likely a predisposing factor for susceptibility to hypertension and/or CAD in an Iranian southeast population.     

A role for ethylene in low-oxygen signaling in rice roots

Summary. Inhibitors of action and synthesis of ethylene (Ag⁺, norbornadien, Co²⁺) were able to reduce the level of γ-aminobutyric acid (Gaba) in rice roots during the development of an anaerobic environment. The inhibitory effect was reversed by the addition of the G protein activator 5′-guanylylimidodiphosphate. Gaba accumulation was modulated by the presence of CO₂ (inhibitor of ethylene action and synthesis) and stimulated by 2-chloroethylphosphonic acid (ethefon). These findings are consistent with a role of ethylene during a low-oxygen stress.     

A role for Fringe in segment morphogenesis but not segment formation in the grasshopper, Schistocerca gregaria

Studies of somitogenesis in vertebrates have identified a number of genes that are regulated by a periodic oscillator that patterns the pre-somitic mesoderm. One of these genes, hairy, is homologous to a Drosophila segmentation gene that also shows periodic spatial expression. This, and the periodic expression of a zebrafish homologue of hairy during somitogenesis, has suggested that insect segmentation and vertebrate somitogenesis may use similar molecular mechanisms and possibly share a common origin. In chicks and mice expression of the lunatic fringe gene also oscillates in the presomitic mesoderm. Fringe encodes an extracellular protein that regulates Notch signalling. This, and the finding that mutations in Notch or its ligands disrupt somite patterning, suggests that Notch signalling plays an important role in vertebrate somitogenesis. Although Notch signalling is not known to play a role in the formation of segments in Drosophila, we reasoned that it might do so in other insects such as the grasshopper, where segment boundaries form between cells, not between syncytial nuclei as they do in Drosophila. Here we report the cloning of a single fringe gene from the grasshopper Schistocerca. We show that it is not detectably expressed in the forming trunk segments of the embryo until after segment boundaries have formed. We conclude that fringe is not part of the mechanism that makes segments in Schistocerca. Thereafter it is expressed in a pattern which shows that it is a downstream target of the segmentation machinery and suggests that it may play a role in segment morphogenesis. Like its Drosophila counterpart, Schistocerca fringe is also expressed in the eye, in rings in the legs, and during oogenesis, in follicle cells. Received: 14 October 1999 / Accepted: 18 January 2000