Nitric oxide, gravity response, and a unified schematic of plant signaling.

Plant signaling components are often involved in numerous processes. Calcium, reactive oxygen species, and other signaling molecules are essential to normal biotic and abiotic responses. Yet, the summation of these components is integrated to produce a specific response despite their involvement in a myriad of response cascades. In the response to gravity, the role of many of these individual components has been studied, but a specific sequence of signals has not yet been assembled into a cohesive schematic of gravity response signaling. Herein, we provide a review of existing knowledge of gravity response and differential protein and gene regulation induced by the absence of gravity stimulus aboard the International Space Station and propose an integrated theoretical schematic of gravity response incorporating that information. Recent developments in the role of nitric oxide in gravity signaling provided some of the final contextual pillars for the assembly of the model, where nitric oxide and the role of cysteine S-nitrosation may be central to the gravity response. The proposed schematic accounts for the known responses to reorientation with respect to gravity in roots-the most well studied gravitropic plant tissue-and is supported by the extensive evolutionary conservation of regulatory amino acids within protein components of the signaling schematic. The identification of a role of nitric oxide in regulating the TIR1 auxin receptor is indicative of the broader relevance of the schematic in studying a multitude of environmental and stress responses. Finally, there are several experimental approaches that are highlighted as essential to the further study and validation of this schematic.

TRP Channels in Insect Stretch Receptors as Insecticide Targets.

Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

Decorin and colchicine as potential treatments for post-haemorrhagic ventricular dilatation in a neonatal rat model.

BACKGROUND: Post-haemorrhagic ventricular dilatation (PHVD) after intraventricular haemorrhage (IVH) remains a significant problem in preterm infants. Due to serious disadvantages of ventriculoperitoneal shunt dependence, there is an urgent need for non-surgical interventions. Considerable experimental and clinical evidence implicates transforming growth factor ß (TGFß) in the pathogenesis of PHVD. Colchicine and decorin are both compounds with anti-TGFß properties. The former downregulates TGFß production and is in clinical use for another fibrotic disease, and the latter inactivates TGFß. OBJECTIVES: We hypothesized that administration of decorin or colchicine, which both have anti-TGFß properties, would reduce ventricular dilatation in a model of PHVD. METHODS: 142 rat pups underwent intraventricular blood injection on postnatal days (PN) 7 and 8. Sixty-nine pups were randomized to colchicine 20 and 50 µg/kg/day or water by gavage for 13 days. Seventy were randomized to decorin 4 mg/kg or saline by intraventricular injection on PN8 and PN13. At PN21, the ventricular area was measured on coronal brain sections. Negative geotaxis was tested at PN14 in controls and in the decorin study group. RESULTS: Ventricular size was not different between animals receiving either drug or water/saline. Intraventricular blood impaired neuromotor performance, but decorin had no effect. CONCLUSION: Two drugs that block TGFß by different mechanisms do not reduce ventricular dilatation in this model. Together with our previous work on losartan and pirfenidone, we conclude that blocking TGFß alone does not prevent the development of PHVD.

Halotolerance is enhanced in carrot callus by sensing hypergravity: influence of calcium modulators and cytochalasin D.

Carrot callus was centrifuged at 10 g and compared to callus growing at 1 g on agar in the presence of increasing sodium chloride concentrations. Growth after 14 days was enhanced in the centrifuged samples versus samples kept at 1 g. This effect was not found when the samples were grown on potassium chloride. At 50 mM NaCl, the calcium ionophore ionomycin was applied to centrifuged and noncentrifuged callus samples. In both experiments, the growth of callus increased with increasing ionomycin concentrations but under 10 g this increase was more enhanced. As inhibitors of calcium influx, lanthanum and gadolinium chloride were chosen in the presence of 50 mM NaCl. Both inhibitors inhibited growth at 1 g at low concentrations of around 2 microM, whereas the centrifuged samples were not or much less so inhibited. We tested an involvement of actin by application of cytochalasin D to callus grown in the presence of 50 mM NaCl. In both types of samples, growth at 1 g and growth at 10 g, cytochalasin D enhanced growth but the effect was clearly stronger at 10 g than at 1 g. As increased halotolerance was only observed in the presence of increased sodium ions, not potassium ions, and as halotolerance is known to be induced by an influx of calcium, the data suggest that a calcium influx induced by hypergravity and possibly modulated by actin caused the observed increase in halotolerance at 10 g.

The onset of gravisensitivity in the embryonic root of flax.

Vertical orientation of emerging roots typically is the first response of plants to gravity. Although root gravitropism has been studied extensively, no conclusive data on the onset of gravisensing exist. We determined the inception of gravisensitivity in flax (Linum usitatissimum) roots by clinorotating germinating seeds after various periods of static orientation (gravistimulation) of imbibed seeds. Gravitropic competency was established about 8 h after imbibition, 11 h prior to germination. The time was determined based on 50% of the newly emerged roots curving in the direction of the gravity vector during static imbibition, despite subsequent clinorotation. The threshold value was affected by the orientation of the seeds. Upward orientation of the micropyle/radicle reduced the number of graviresponding roots to about one-half. Prolonged clinorotation weakened the graviresponse. Gravisensing was accompanied by the development of amyloplasts, but the actin cytoskeleton was not involved because imbibition in Latrunculin B did not affect the onset of gravisensitivity or germination, and the development of F-actin in untreated controls was observed only after the onset of gravisensitivity.

Developmental and behavioral effects of acrylamide in Fischer 344 rats.

Human exposures to acrylamide (ACR), a known neurotoxicant, can occur via a variety of substances, including cigarette smoke and the ingestion of certain carbohydrate-based foods cooked at high temperatures. In this study, Fischer 344 sperm plug-positive female rats were treated daily with ACR (0, 0.5, 1.0, 2.5, 5.0 or 10.0 mg/kg/day) by gavage beginning on gestation day 7. Dosing of dams ended when litters were born; pups received daily gavage at the same dose as their dam from postnatal day (PND) 1 through PND22. Pups were tested using a battery of behavioral assessments from PNDs 4-22. Statistically significant decreases in body weight were observed in pups exposed to ACR at doses as low as 1.0 mg/kg/day (treatmentxday; repeated measures ANOVA, P<0.0001). No statistically significant differences among treatment groups were observed in righting reflex, forelimb hang, or open field measures of activity. Statistically significant effects of ACR were observed at the 10 mg/kg/day dose on negative geotaxis performance (P<0.01) and a linear trend in fall-time latencies on Rotarod performance on PNDs 21-22 (P<0.05), with higher doses producing shorter latencies. These results suggest that ACR exposure produces deficits in development and motor coordination that are observable before weaning.

Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem.

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament-disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts.

Fluoxetine facilitates use-dependent excitability of human primary motor cortex.

OBJECTIVES: In poststroke patients, fluoxetine, a selective serotonin-reuptake inhibitor, as an adjunct to physical therapy provided a better functional recovery from motor deficits. The aim of this study was to investigate the effect of a single dose of 20 mg fluoxetine on motor learning and associated cortical changes in healthy right-handed subjects in order to get deeper insight into its facilitating influence on human motor cortex. METHODS: Subjects performed a motor task consisting of a simultaneous co-contraction of the abductor pollicis brevis (APB) and the deltoid muscle with and without fluoxetine in a placebo-controlled double-blinded crossover study design. Immediately before and after motor learning motor output maps of the APB muscle were assessed in order to get insight into plastic changes of the muscle representation. RESULTS: We found a significantly improved motor performance under both conditions without having substantial differences between placebo and fluoxetine. After the completion of the motor task there was a medial shift of the APB muscle motor output map. Only after the administration of fluoxetine the sum of MEP amplitudes (SOA) increased and the motor output map enlarged. CONCLUSIONS: These findings provide evidence for a use-dependent facilitating effect of fluoxetine on cortical excitability but not on motor performance. SIGNIFICANCE: Our findings are not in line with previous experiments in poststroke patients. However, long-term treatment with fluoxetine may additionally improve motor function by upregulating serotonergic receptors. Further studies investigating the influence of long-term treatment on cortical excitability and psychophysics may therefore provide deeper insight into a possible therapeutical efficiency of fluoxetine in poststroke patients.

Perception mechanism of gravity stimuli in hypergravity-induced growth inhibition of azuki bean roots.

We reported that elongation growth of plant shoots and roots is suppressed by hypergravity, with the rate decreasing in proportion to logarithm of the magnitude of gravity. In hypergravity-induced growth inhibition of shoots, graviperception is supposed to be independent of that in gravitropism and to involve mechanoreceptors. However, the graviperception mechanism in the hypergravity-induced growth inhibition of roots is not known. In the present study, we compared the mechanism in the hypergravity-induced growth inhibition of roots with that in gravitropism. The removal of root cap did not influence hypergravity-induced growth inhibition of roots, although the gravitropic curvature was completely inhibited. Hypergravity had no effects on growth of azuki bean roots in the presence of lanthanum or gadolinium, which are blockers of mechanoreceptors. On the contrary, lanthanum or gadolinium at the same concentration did not influence gravitropism of roots. These results suggest that the graviperception mechanism in the hypergravity-induced growth inhibition of roots is independent of that in gravitropism. Hypergravity-induced growth inhibition of azuki bean roots was observed irrespective of the direction of stimuli, which disappeared in the presence of lanthanum or gadolinium. Thus, in the hypergravity-induced growth inhibition, roots may perceive the gravity signal by mechanoreceptors on the plasma membrane independently of the direction of stimuli, and may utilize it to regulate their growth rate.

Gravitactic signal transduction elements in Astasia longa investigated during parabolic flights.

Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitactic behavior. Many experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism. The goal of the present study was to examine elements in the sensory transduction by means of inhibitors of gravitaxis and the intracellular calcium concentration during short microgravity periods. During the course of six parabolic flights (ESA 31th parabolic flight campaign and DLR 6th parabolic flight campaign) the effects of trifluoperazine (calmodulin inhibitor), caffeine (phosphodiesterase inhibitor) and gadolinium (blocks mechano-sensitive ion channels) was investigated. Due to the extreme parabolic flight maneuvers of the aircraft alternating phases of 1.8 x g(n) (about 20 s) and microgravity (about 22 s) were achieved (g(n): acceleration of Earth's gravity field). The duration of the microgravity periods was sufficient to detect a loss of cell orientation in the samples. In the presence of gadolinium impaired gravitaxis was found during acceleration, while caffeine-treated cells showed, compared to the controls, a very precise gravitaxis and faster reorientation in the 1.8 x g(n) period following microgravity. A transient increase of the intracellular calcium upon increased acceleration was detected also in inhibitor-treated samples. Additionally, it was found that the cells showed a higher calcium signal when they deviated from the vertical swimming direction. In the presence of trifluoperazine a slightly higher general calcium signal was detected compared to untreated controls, while gadolinium was found to decrease the intracellular calcium concentration. In the presence of caffeine no clear changes of intracellular calcium were detected compared to the control.

Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton.

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

Carbaryl-induced behavioural and reproductive abnormalities in the earthworm Metaphire posthuma: a sensitive model.

Carbaryl, an N-methyl carbamate insecticide, is used in India to control foliar insects, but, due to soil contamination, it also adversely affects non-target organisms such as earthworms. This paper deals with the toxic effects of carbaryl on the behavioural and reproductive profiles of the earthworm, Metaphire posthuma. Locomotion and geotaxis were significantly affected, even after a 20-minute exposure to 0.125ppm carbaryl. The hatching of cocoons was altered at 0.5ppm, whereas cocoon production was retarded even at 0.125ppm carbaryl. No cocoon production was observed at 2.0ppm carbaryl. Sperm head abnormalities were reported even at the lowest test concentration of 0.125ppm. Wavy head abnormalities were observed at 0.125ppm carbaryl, whereas at 0.25ppm and 0.5ppm, the sperm heads became amorphous and the head nucleus was turned into granules deposited within the wavy head. It is concluded that the earthworm could be used as an ecosystem model for the initial toxicity testing of environmental pollutants.

Tip-growing cells of the moss Ceratodon purpureus Are gravitropic in high-density media.

Gravity sensing in plants and algae is hypothesized to rely upon either the mass of the entire cell or that of sedimenting organelles (statoliths). Protonemata of the moss Ceratodon purpureus show upward gravitropism and contain amyloplasts that sediment. If moss sensing were whole-cell based, then media denser than the cell should prevent gravitropism or reverse its direction. Cells that were inverted or reoriented to the horizontal displayed distinct negative gravitropism in solutions of iodixanol with densities of 1.052 to 1.320 as well as in bovine serum albumin solutions with densities of 1.037 to 1.184 g cm(-3). Studies using tagged molecules of different sizes and calculations of diffusion times suggest that both types of media penetrate through the apical cell wall. Estimates of the density of the apical cell range from 1.004 to 1.085. Because protonemata grow upward when the cells have a density that is lower than the surrounding medium, gravitropic sensing probably utilizes an intracellular mass in moss protonemata. These data provide additional support for the idea that sedimenting amyloplasts function as statoliths in gravitropism.

Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli.

Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.

The gravitropic setpoint angle of dark-grown rye seedlings and the role of ethylene.

The orientation growth of coleoptiles of dark-grown seedlings of rye (Secale cereale L. cv. Marder II), when grown under various conditions, was analysed with respect to the gravivector ('gravitropic setpoint angle', GSA). Coleoptiles growing through moist vermiculite attain and maintain a GSA with an average of about 180 degrees, i.e. a vertical orientation. Seedlings growing uncovered either on the surface of vermiculite or positionally fixed on filter paper attain and maintain a GSA of 140-150 degrees (i.e. deviating from the vertical by an average of 30-40 degrees ). Changing the position of the embryo relative to the horizontally fixed seed kernel or of the angle of the seed with respect to gravity during germination (+/-40 degrees relative to the horizontal) had no significant effect on the subsequent GSA of both covered and uncovered seedlings. The GSA of uncovered coleoptiles could be restored close to 180 degrees by treatment of the seedlings with ethylene, either applied via ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC) as well as by fruit-released ethylene. The results are discussed with respect to the mechanism of the regulation of gravitropic growth of grass seedlings.

Protection of negative gravitaxis in Euglena gracilis Z against gamma-ray irradiation by Trolox C.

The protective effects of Trolox on the inhibition of negative gravitaxis in Euglena gracilis exposed to 200 Gy 60Co gamma-rays were examined using different concentrations (1, 10 and 100 microM). The orientation precision of the negative gravitaxis was quantified using the r-value. A significant decrease in the r-value was observed in gamma-irradiated samples (0.18+/-0.03) compared to those of non-irradiated samples (0.47+/-0.03). There were no significant changes in the r-value of cells exposed to 200 Gy gamma-rays by the addition of 1 or 10 microM of Trolox. A significant increase (0.19) in the r-value of cells exposed to 200 Gy with 100 microM Trolox was observed. The results indicates that Trolox at a concentration of 100 microM protects negative gravitaxis against 60Co gamma-ray irradiation at a dose of 200 Gy. It also suggests that the negative gravitaxis of Euglena gracilis is affected by free radicals.

Gravicurvature loss, changes in ultrastructure and calcium balance of pea root statocytes treated with EGTA.

Calcium is known to play a key role in the regulation of a wide variety of cellular events in plants, in particular as a second messenger in the transduction of gravity signals in statocytes. In order to investigate the role of extracellular calcium in gravity perception, many investigators have used ethylene glycol-bis-(aminoethyl ether)-N,N'-tetraacetic acid (EGTA), whose main property is the ability to form stable water-soluble complex compounds with divalent metals, particularly with Ca2+ ions, that do not penetrate across plasma membrane into a cell.

Interactions between N-methyl-D-aspartate and nitric oxide in the modulation of ultrasonic vocalizations of infant rats.

The effects of interactions between N-methyl-D-aspartate (NMDA) and nitric oxide on ultrasonic vocalizations, motor activity and body temperature was investigated in 9-10-day-old rat pups. The competitive NMDA receptor antagonist, 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and the nitric oxide synthase inhibitor, nitro-L-arginine-methyl ester (L-NAME), decreased the emission of ultrasonic vocalizations while NMDA and the nitric oxide precursor, L-arginine, produced a trend toward increased emission of ultrasonic vocalizations. CCP also attenuated the geotaxic response. Co-administration of CPP with L-NAME virtually abolished the emission of ultrasonic vocalizations and the ability to show the geotaxic response while co-administration of NMDA with L-arginine increased the emission of ultrasonic vocalizations and decreased body temperature with no effect on the geotaxic response. NMDA and L-arginine reversed the effects of L-NAME, but not of CPP, on ultrasonic vocalizations. L-arginine but not NMDA antagonized the effect of CPP on the geotaxic response. Our results confirmed the functional coupling between NMDA receptor activation and nitric oxide in modulating anxiety-like behavior and motor coordination in infant rats.

Effects of light on gravitaxis and velocity in Chlamydomonas reinhardtii.

The effects of light on gravitaxis and velocity in the bi-flagellated green alga Chlamydomonas reinhardtii were investigated using a real time automatic tracking system. Three distinct light effects on gravitaxis and velocity with parallel kinetics were found. Photosynthetically active continuous red light reversibly enhances the swimming velocity and increases or decreases the precision of gravitaxis, depending on its initial level. Blue light flashes induce fast transient increases in velocity immediately after the photophobic response, and transiently decrease or even reverse negative gravitaxis. The calcium dependence of this response, its fluence-response curve and its spectral characteristics strongly suggest the participation of chlamy-rhodopsin in this effect. The third response, a prolonged activation of velocity and gravitaxis, is also induced by blue light flashes, which can be observed even in calcium-free medium.