Effect of ion channel blockers and H<Superscript>+</Superscript>-ATPase inhibitors on generation of local electrical response in a cucumber leaf

Electric potential difference was measured with extracellular electrodes between the leaf surface of 2-week-old cucumber (Cucumis sativus L.) plants and soil solution. When the leaf region with a diameter of 5 mm was gradually cooled during a 105-s period to 8–9°C, the temperature drop induced a local (confined to the cooled area) nonpropagating pulse-wise electric activity. The cessation of cooling was followed by gradual (within 12–15 min) restoration of the initial potential difference. Two peaks of electric potential with amplitudes of 100–120 mV usually appeared upon cooling. The first depolarizing stage of the pulse activity was sensitive to inhibition of voltage-gated and mechanosensitive calcium channels of plasmalemma by lanthanum and gadolinium chlorides and to verapamil treatment. Furthermore, the inhibition of this stage by ruthenium red implies the release of calcium ions from intracellular stores. The initial slow depolarization was followed by a fast depolarizing shift, which was sensitive to La³⁺ and the anion channel inhibitor 4-acetamido-4′-isothiocyano-stilbene-2,2′-dilsulfonic acid. At the next stage repolarization developed, which was sensitive to potassium channel blockers, tetraethylammonium and quinine sulfate. The influence of ion channels blockers indicates that generation of local bioelectric response is based on fluxes of the same ion species that are involved in the action potential. The depolarization stage was due to the transient Ca²⁺ influx into the cytosol from the apoplast and intracellular stores, together with the anion efflux from the cell; the repolarization stage involved potassium ions. Both stages of electric pulse generation were retarded by the H⁺-ATPase inhibitors, sodium orthovanadate and dicyclohexylcarbodiimide, which implies the involvement of the proton pump in the origin of electric pulses examined.     

Dissection of heat-induced systemic signals: superiority of ion fluxes to voltage changes in substomatal cavities

Using non-invasive ion-selective microprobes, that were placed in substomatal cavities, long-distance signalling has been investigated in intact Hordeum vulgare and Vicia faba seedlings. Heat (flame), applied to one leaf (S-leaf), triggers apoplastic ion activity (pH, pCa, pCl) transients in a distant leaf (T-leaf), all largely independent of simultaneously occurring action potential-like voltage changes. While apoplastic pCa and pH increase (Ca²⁺-, H⁺-activities decrease), pCl decreases (Cl⁻-activity increases). As the signal transfer from the S- to the T-leaf is too fast to account for mass flow, the heat-induced pressure change is primarily responsible for changes in voltage (H⁺ pump deactivation) as well as for the ion fluxes. The pCa transient precedes the pCl- and pH responses, but not the voltage change. Since the apoplastic pCl decrease (Cl⁻ increase) occurs after the pCa increase (Ca²⁺ decrease) and after the depolarization, we argue that the Cl⁻ efflux is a consequence of the Ca²⁺ response, but has no part in the depolarization. Kinetic analysis reveals that pH- and pCl changes are interrelated, indicated by the action of the anion channel antagonist NPPB, which inhibits both pCl- and pH changes. It is suggested that efflux of organic anions into the apoplast causes the pH increase rather than the deactivation of the plasma membrane H⁺ pump. Since there is considerably more information in ion activity changes than in a single action- or variation potential and heat-induced ion fluxes occur more reliably than voltage changes, released by milder stimuli, they are considered systemic signalling components superior to voltage.     

Salinity effect on plant growth and leaf demography of the mangrove, Avicennia germinans L.

We assessed the effect of salinity on plant growth and leaf expansion rates, as well as the leaf life span and the dynamics of leaf production and mortality in seedlings of Avicennia germinans L. grown at 0, 170, 430, 680, and 940 mol m⁻³ NaCl. The relative growth rates (RGR) after 27 weeks reached a maximum (10.4 mg g⁻¹ d⁻¹) in 170 mol m⁻³ NaCl and decreased by 47 and 44% in plants grown at 680 and 940 mol m⁻³ NaCl. The relative leaf expansion rate (RLER) was maximal at 170 mol m⁻³ NaCl (120 cm m⁻² d⁻¹) and decreased by 57 and 52% in plants grown at 680 and 940 mol m⁻³ NaCl, respectively. In the same manner as RGR and RLER, the leaf production (P) and leaf death (D) decreased in 81 and 67% when salinity increased from 170 to 940 mol m⁻³ NaCl, respectively. Since the decrease in P with salinity was more pronounced than the decrease in D, the net accumulation of leaves per plant decreased with salinity. Additionally, an evident increase in annual mortality rates (λ) and death probability was observed with salinity. Leaf half-life (t _0.5) was 425 days in plants grown at 0 mol m⁻³ NaCl, and decreased to 75 days at 940 mol m⁻³ NaCl. Thus, increasing salinity caused an increase in mortality rate whereas production of new leaves and leaf longevity decreased and, finally, the leaf area was reduced.     

Effect of Cold Osmotic Shock on K+ Efflux from Nicotiana tabacum L. Leaf Discs Induced by Abscisic Acid and Ionophores

Cold osmotic shock, i.e., transfer of tobacco (Nicotiana tabacumL. cv. Samsun NN) leaf discs from 0.5 M mannitol at 25°Cto distilled water at 2°C appears to modify the events selectivelyat the plasma membrane of tobacco leaf cells. Especially, theefflux of K+ and ion leakage was markedly increased by coldosmotic shock, which is dependent on both osmotic pressure andthe temperature of the solution bathing the discs; i.e., K+efflux was increased by raising the osmotic pressure of mannitoland further intensified by subsequently lowering the temperature.The shock effects are temporary and disappear 2 to 3 hr afterthe treatment. The efflux of K+ from the discs into the external medium (1mM MES-Tris, pH 6.0) was stimulated by abscisic acid (ABA) andgramicidin S, slightly stimulated by nigericin and slightlyinhibited by kinetin. Valinomycin was insensitive to K+ efflux. ABA-induced stimulation of K+ efflux was inhibited by cold osmoticshock, but not the gramicidin-induced stimulation. The ABA-inducedstimulation was partly inhibited by kinetin, whereas the gramicidin-inducedone was not. The stimulatory effect on K+ efflux was additivewhen ABA and ionophore were added together to the external medium. These results indicate that ABA acts at the plasma membranethat is selectively modified by cold osmotic shock. The stimulationof K+ efflux induced by ABA and ionophore occurs via differentprocesses. (Received December 22, 1980; Accepted May 21, 1981)     

39K nuclear magnetic resonance and a mathematical model of K+ transport in human erythrocytes

³⁹K nuclear magnetic resonance was used to measure the efflux of K⁺ from suspensions of human erythrocytes [red blood cells (RBCs)], that occurred in response to the calcium ionophore, A23187 and calcium ions; the latter activate the Gárdos channel. Signals from the intra- and extracellular populations of ³⁹K⁺ were selected on the basis of their longitudinal relaxation times, T ₁, by using an inversion- recovery pulse sequence with the mixing time, τ₁, chosen to null one or other of the signals. Changes in RBC volume consequent upon efflux of the ions also changed the T ₁ values so a new theory was implemented to obviate a potential artefact in the data analysis. The velocity of the K⁺ efflux mediated by the Gárdos channel was 1.19±0.40 mmol (L RBC)⁻¹ min⁻¹ at 37°C.     

Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Interaction between photon flux density and elevated temperatures on photoinhibition in Alocasia macrorrhiza

The effects of light and elevated temperatures on the efficiency of energy conversion in PSII [?_PSII = (Fm′−Fs)/Fm′], pigment composition and heat tolerance of shade-acclimated Alocasia macrorrhiza were investigated. Leaf discs were exposed for 3 h to high light (HL; 1600 μmol photons · m⁻² · s⁻¹) or low light (LL; 20 μmol photons · m⁻² · s⁻¹) and a series of constant temperatures ranging from 30 to 49 °C. All HL treatments led to rapid and severe decreases in ?_PSII. During the 2-h recovery period (LL, 25 °C) following the HL treatments, fast and slow recovery phases could be distinguished. Leaf discs that had experienced HL and 30 °C recovered completely while no recovery of ?_PSII was seen after a 3-h exposure to HL and 45 °C. A 3-h exposure to 45 °C at LL led to a less severe decrease in ?_PSII and complete recovery was accomplished after less than 1 h. Under LL conditions a temperature of 49 °C was necessary to cause an irreversible decrease in ?_PSII, followed by necrosis the next day. Streptomycin had no effect on the degree of reduction and recovery in ?_PSII discs exposed to HL and 35–45 °C, but partially inhibited recovery in discs exposed to HL and 30 °C. Streptomycin led to a more severe decrease in ?_PSII at LL and 49 °C and completely inhibited recovery. Streptomycin had no effect on the conversion of the xanthophyll-cycle pigments during the treatment or the recovery. The epoxidation state was roughly the same in all leaf discs after a 3-h HL treatment (0.270–0.346) irrespective of the exposure temperature. The back-conversion of zeaxanthin into violaxanthin after a 2-h recovery period was only seen in leaf discs that had been exposed to HL and 30 °C. The thermotolerance of shade A. macrorrhiza leaves of 49.0 ± 0.7 °C (determined by fluorescence) coincided with the temperature at which damage occurred in leaf discs exposed to LL. However, under HL the critical temperature under which necrosis occurred was much lower (42 °C). The thermotolerance of A. macrorrhiza shade leaves could be increased by a short exposure (<20 min) to slightly elevated temperatures. Received: 11 June 1997 / Accepted: 9 September 1997     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

Reactive oxygen species and imbalance of calcium homeostasis contributes to curcumin induced programmed cell death in Leishmania donovani

Curcumin, a polyphenol compound, has been recognized as a promising anti-cancer drug. The purpose of the present study was to investigate the cytotoxicity of curcumin to Leishmania donovani, the causative agent for visceral leishmaniasis. Flow cytometric analysis revealed that curcumin induced cell cycle arrest at G2/M phase. Incubation of Leishmania promastigotes with curcumin caused exposure of phosphatidylserine to the outer leaflet of plasma membrane. This event is preceded by curcumin-induced formation of reactive oxygen species (ROS) and elevation of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (ΔΨm), release of Cytochrome c into the cytosol and concomitant nuclear alterations that included deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) and DNA fragmentation. Taken together, these data indicate that curcumin has promising antileishmanial activity that is mediated by programmed cell death and, accordingly, merits further investigation as a therapeutic option for the treatment of leishmaniasis.     

Storage of Porcine Articular Cartilage at High Subzero Temperatures

Objective: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. Design: 10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (−10, −15, and −20 °C), and held for 30 min. After warming, 70 μm slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. Results: Results demonstrated excellent cell recovery (>75%) at −10°C provided ice did not form. Excellent cell recovery (>70%) occurred at −15°C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at −20°C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form Conclusions: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.     

Leaf water characteristics and drought acclimation in sunflower genotypes

The responses of leaf water parameters to drought were examined using three sunflower (Helianthus annuus L.) genotypes. Osmotic potential at full water saturation (π¹⁰⁰), apoplastic water fraction (AWF) and bulk elastic modulus (BEM) were determined by pressure-volume curve analysis on well watered or on water-stressed plants (−1.0 MPa Ψ₁ < −1.5 MPa) previously drought-pretreated or not. The drought-pretreated plants were subjected to a 7-day drought period (predawn leaf water potential reached −0.9 MPa) followed by 8 days of rewatering. In well watered plants, all genotypes in response to drought acclimation displayed a significantly decreased π¹⁰⁰ associated with a decrease in the leaf water potential at the turgor-loss point (decrease in Ψ_tlp was between 0.15 and 0.21 MPa, depending on the genotype). In two genotypes, drought acclimation affected the partitioning of water between the apoplastic and symplastic fractions without any effect on the total amount of water in the leaves. As a third genotype displayed no modification of AWF and BEM after drought acclimation, the decreased π¹⁰⁰ was only due to the net accumulation of solutes and was consistent with the adjustment of the photochemical efficiency observed previously in this genotype in response to drought acclimation. In water-stressed plants, the osmotic adjustment (OA) can increase further beyond that observed in response to the drought pretreatment. However, the maintenance of photosynthetic rate and stomatal conductance at low leaf water potentials not only depends on the extent of osmotic adjustment, but also on the interaction between OA and AWF or BEM. Adaptative responses of leaf water parameters to drought are thus quite contrasted in sunflower genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photooxidative damage to the photosynthetic apparatus during chilling

Short periods of chilling in the presence of light (up to 6 h: 1°C; 270 W/m²) decreased the subsequent apparent photosynthesis and chlorophyll fluorescence of leaf discs of Cucumis sativus L. cv. Kleine Groene Scherpe. The extent of the injury depended on the duration of the chilling pretreatment. After 6 h the subsequent apparent photosynthesis even reached a negative value, and it increased only slightly during the next 2 1/2 h. The decrease of apparent photosynthesis was not a consequence of increased dark respiration but was of photooxidative origin since the presence of both light and oxygen was required. Preincubation in the light for 2 h at 20°C sensitized leaf discs to subsequent photooxidation during chilling. Prompt and delayed chlorophyll fluorescence decreased simultaneously after chilling and light treatment. The corresponding decrease of photosynthesis and chlorophyll fluorescence is discussed in relation to primary photooxidative damage to the photosystems in the chloroplast membrane.     

The influence of temperature and nitrogen source on growth and nitrogen uptake of two polar-desert species,Saxifraga caespitosa and Cerastium alpinum

Polar-desert plants experience low average air temperatures during their short growing season (4–8 °C mean July temperature). In addition, low availability of inorganic nitrogen in the soil may also limit plant growth. Our goals were to elucidate which N sources can be acquired by polar-desert plants, and how growth and N-uptake are affected by low growth temperatures. We compared rates of N-uptake and increases in mass and leaf area of two polar-desert species (Cerastium alpinum L. and Saxifraga caespitosa L.) over a period of 3 weeks when grown at two temperatures (6 °C vs. 15 °C) and supplied with either glycine, NH₄ ⁺ or NO₃ ⁻. At 15 °C, plants at least doubled their leaf area, whereas there was no change in leaf area at 6 °C. Measured mean N-uptake rates varied between 0.5 nmol g⁻¹ root DM s⁻¹ on glycine at 15 °C and 7.5 nmol g⁻¹ root DM s⁻¹ on NH₄ ⁺ at 15 °C. Uptake rates based upon increases in mass and tissue N concentrations showed that plants had a lower N-uptake rate at 6 °C, regardless of N source or species. We conclude that these polar-desert plants can use all three N sources to increase their leaf area and support flowering when grown at 15 °C. Based upon short-term (8 h) uptake experiments, we also conclude that the short-term capacity to take up inorganic or organic N is not reduced by low temperature (6 °C). However, net N-uptake integrated over a three-week period is severely reduced at 6 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Systemic signalling in barley through action potentials

Using apoplastic voltage- and ion selective microprobes, in barley leaves action potentials (APs) have been measured, which propagate acropetally as well as basipetally from leaf to leaf or from root to leaf following the application of mild salt stress (e.g. 30–50 mM KCl or NH₄Cl) or amino acids (e.g. 1 mM glutamic acid or 5 mM GABA). Voltage changes were biphasic, followed an ‘all-or-none’ characteristic, and propagated at 20–30 cm min⁻¹ irrespective of the direction. With the salt-induced APs, a strong initial depolarization is the main AP-releasing factor that first causes Ca²⁺ influx and then anion efflux. Ca²⁺ influx coincides with an initial slower depolarization, the rapid anion efflux causes the typical voltage ‘break-through’. Subsequently, K⁺-efflux starts after the depolarizing voltage has passed the K⁺ equilibrium potential (inversion of the K⁺ driving force). Glutamic acid and GABA induce APs not through membrane depolarization, but presumably by binding to a putative receptor or to ligand-gated Ca²⁺-conducting channels, respectively, followed by Ca²⁺ induced activation of anion efflux. APs are accompanied by transient apoplastic pH increase (about 1 unit), and by cytoplasmic pH decrease (about 0.5 units). The apoplastic pH change is interpreted as an indicator of stress, the cytoplasmic pH change as a prerequisite for defence related gene activation. Since APs are released by agents added in a moderate concentration range, it is suggested that they may serve as first and fast systemic signals following attack from pathogens.     

Effect of Abscisic Acid on the K+ Efflux and Membrane Potential of Nicotiana tabacum L. Leaf Cells

K⁺ efflux from tobacco (Nicotiana tabacum L, cv. Samsun NN)leaf discs into the external medium was increased and the membranepotential (Em) changed in the positive direction with a changein pH from 8.0 to 4.0. E_m was affected by the external concentrationof KCl, greatly decreasing with a change in concentration from1 mM to 100 mM. The equilibrium potential of the membrane forK⁺ (E_k) was decreased in a Nernst fashion with increasing externalconcentrations of KCl. E_k is more positive than E_m above ca.50 µM KCl. Most of the experiments were carried out underconditions in which the difference between the electrochemicalpotential for K⁺ on the inside to the outside of the cell (µ_kis positive. Thus, K⁺ may passively flow to the outside of thecells accompanied by the depolarization of the membrane. Abscisic acid (ABA) increased the K⁺ efflux under conditionsof passive transport. K⁺ efflux was accelerated with an increasingconcentration of ABA, being maximal at 10^–4 M–10^–3M. This acceleration was due to the enhancement of the potassiummotive force (µ_k/F) which is the force causing the netpassive transport of K⁺. The membrane potential was decreasedfrom –205 mV to –170 mV by 2 x 10^–4 M ABAwithin 10 min. The depolarization was not transient, being lostfor at least 3 hr. These results show that ABA accelerated passive K⁺ efflux, whichaccompanied depolarization of the membrane. (Received June 22, 1981; Accepted August 24, 1981)     

Proton efflux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment

Elongation growth of plant cells occurs by stretching of cell walls under turgor pressure when intermolecular bonds in the walls are temporarily loosened. The acid-growth theory predicts that wall loosening is the result of wall acidification because treatments (including IAA and fusicoccin) that cause lowered wall pH cause elongation. However, conclusive evidence that IAA primarily reduces wall pH has been lacking. Calcium has been reported to stiffen the cell walls. We have used a microelectrode ion-flux measuring technique to observe directly, and non-invasively, the net fluxes of protons and calcium from split coleoptiles of oats (Avena sativa L.) in unbuffered solution. Normal net fluxes are 10 nmol · m⁻² · s⁻¹ proton efflux and zero calcium flux. The toxin fusicoccin (1 μM) causes immediate efflux from tissue not only of protons, but also of calcium, about 110 nmol · m⁻² · s⁻¹ in each case. The data fit the “weak acid Donnan Manning” model for ion exchange in the cell wall. Thus we associate the known “acid-growth” effect of fusicoccin with the displacement of calcium from the wall by exchange for protons extruded from the cytoplasm. Application of 10 μM IAA causes proton efflux to increase transiently by about 15 nmol · m⁻² · s⁻¹ with a lag of about 10 min. The calcium influx decreases immediately to an efflux of about 20 nmol · m⁻² · s⁻¹. It appears that auxin too causes an “acid-growth” effect, with extruded protons exchanging for calcium in the cell walls. I. Arif is currently recieving an AIDAB scholarship. This work was supported by an Australian Research Council grant to I.A. Newman.     

Unique after-hyperpolarization accompanying action potential inChara globularis

A unique after-hyperpolarization was found in internodal cells ofChara globularis. The cells generated an ordinary action potential due to regenerative depolarization induced by the outward electric current pulse larger than a threshold stimulus. After reaching a depolarizing peak, the membrane potential repolarized and overshooted the resting potential to a value which was somehow 40 mV more negative than the resting potential before stimulation (after-hyperpolarization). Since the membrane resistance increased during the after-hyperpolarization, the after-hyperpolarization is thought to be caused by an increase in the resistance (decrease in the conductance) of the passive diffusion channel.     

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l⁻¹) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l⁻¹) + thidiazuron (1 mg l⁻¹). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wind tunnel assays of olfactory responses of femaleRhagoletis pomonella flies to apple volatiles: effect of wind speed and odour release rate

Little is known about how adults of the corn leafhopper,Dalbulus maidis (DeLong & Wolcott), and its congeners survive subfreezing temperatures at high elevations during the dry winter in Mexico. In the laboratory, duration of survival at −5°C was measured for four MexicanDalbulus species:D. maidis, D. elimatus (Ball),D. gelbus DeLong andD. quinquenotatus DeLong & Nault; and a closely related North American species,Baldulus tripsaci Kramer & Whitcomb. Adult leafhoppers reared under environmental conditions that simulated the beginning of the dry winter season during October in Mexico (‘October-reared’) were at least twice as tolerant of −5°C than adults reared under environmental conditions that simulated the beginning of the wet summer season during June (‘June-reared’).Dalbulus species found primarily at high elevations, such asD. elimatus, were seven times more tolerant of −5°C thanD. quinquenotatus, a species which overwinters at low to mid elevations on itsTripsacum hosts. October-rearedD. maidis adults survived relatively short periods at −5°C (LT₅₀=8.9h) compared to October-rearedD. elimatus adults (LT₅₀=42.3h). This suggests that in Mexico,D. maidis either overwinters in protected habitats at higher elevations or it migrates to lower, frost-free regions. October-rearedB. tripsaci adults, which overwinter in the egg stage, were intolerant of −5°C (LT₅₀=2.6h). A conditioning period for 1 h at +5°C before and after exposure to −5°C significantly improved survival forD. maidis. Supercooling points (SCPs) were between −23 and −20°C, indicating that mortality of these leafhoppers at −5°C was due to cold shock injury rather than internal ice formation.     

A Relationship between Membrane Permeability and Ethylene Production at High Temperature in Leaf Tissue of Phaseolus vulgaris L.

Leaf discs cut from primary leaves of Phaseolus vulgaris L cvMasterpiece were incubated at temperatures higher than the growthtemperature of 25 °C Both basal and wound ethylene productionincreased up to temperatures of 35–37 5 °C, thereafterdeclining rapidly There was no detectable ethylene productionat temperatures above 42 5 °C Exposure of leaf discs tohigh temperature for 60 mm resulted in a large production ofwound ethylene when they were returned to 25 °C The magnitudeof ethylene production was related to the initial incubationtemperature as was the length of the lag period before maximumproduction was achieved The results are discussed in relationto the requirement for continued membrane integrity for ethyleneproduction ethylene, temperature, membrane permeability, Phaseolus vulgaris L, dwarf bean