Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Cytosolic Ca2+ pulses and protein kinase activation in the signal transduction pathways leading to the plant oxidative burst

The oxidative burst, the rapid production of O₂- and H₂O₂ by plant cells in response to pathogens and Stressors, is a critical step in plant disease resistance and is controlled by several different elicitor-initiated signaling pathways. While different defense elicitors appear to activate disparate initial steps in signaling the oxidative burst, all of the elicitors tested thus far appear to stimulate pathways that converge on the same three core signaling intermediates: 1) the Ca₂₊-independent activation of a mitogen-activated protein kinase (MAPK) family member, 2) the influx of Ca₂₊ into the cytosol, deriving most critically from an internal compartment, and 3) the Ca²⁺-dependent activation of additional protein kinases including a second MAPK homologue and possibly calcium dependent protein kinases (CDPKs). Data from several recent reports are summarized to place these signaling events into a complete and updated model of signaling to the plant oxidative burst.     

Evidence for a Mechanically Induced Oxidative Burst

Rapid release of H2O2 may constitute an initial defense response mounted by a plant. Inauguration of this oxidative burst is known to occur upon stimulation with chemical elicitors, but the possibility of mechanical elicitation arising from pathogen penetration/weakening of the cell wall has never been examined. To introduce an adjustable mechanical stress on the plasma membrane, cultured soybean (Glycine max Merr. cv Kent) cells were subjected to defined changes in medium osmolarity. Dilution of the medium with water or resuspension of cells in sucrose solutions of reduced osmolarity yielded an oxidative burst similar to those stimulated by chemical elicitors. Furthermore, the magnitude of oxidant biosynthesis and osmotic stress correlated directly. Upon return of the cells to normal tonicity, the oxidative burst abruptly halted, indicating that its expression depended on maintenance of the osmotic stress and not on any external chemical signal. To confirm the ability of soybean cells to respond to a mechanical stimulus with induction of an oxidative burst, cells were subjected to direct physical pressure. Application of pressure yielded a characteristic oxidative burst. Because neither these cells nor those subjected to osmotic pressure were damaged by their treatments, we conclude that plant cells can detect mechanical disturbances and initiate a classical defense reaction in response.     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O₂^•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O₂^•− in a hydrogen peroxide (H₂O₂)-dependent manner. However, this model is incomplete since the source of the initially required H₂O₂ could not be explained. Based on the recently proposed role for H₂O₂-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.     

12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis

 In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L.) Heynh. The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids. The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A. thaliana. Substrate conversion was analyzed using a stereospecific assay. The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid [K _m = 35 μM, V _max 53.7 nkat (mg protein)⁻¹] to the corresponding 3-2(2′(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3. Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis. Received: 21 October 1999 / Accepted: 10 December 1999     

Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family

Molecular characterization has been accomplished for five members of the peroxidase gene family in French bean. The most important of these, designated FBP1, corresponds to the isoform believed to be responsible for the apoplastic oxidative burst demonstrated by suspension-cultured cells in response to fungal elicitor. Identification was made by a complete match of six peptide sequences derived from the native protein to the translated sequence of the cDNA. Modelling of the surface structure in comparison with two other members of the peroxidase family did not reveal any unusual features which might account for its role in the oxidative burst. However, FBP1 when expressed in Pichia pastoris generated H₂O₂ using cysteine at pH 7.2, a specific property of the native protein when isolated from suspension-cultured cells. FBP1, together with other members of the family, were all induced in cell cultures by elicitor action although they all showed some expression in non-induced cultured cells. They were also expressed in all tissues examined with varying levels of intensity of detection in northern blots. This was confirmed by in situ hybridization and FBP1 expression was confirmed in tissues where it has been previously detected by immunolocalization methods. Assigning roles to individual peroxidases is an important goal and molecular identification of the oxidative burst peroxidase allows further exploration of the relative roles of the different systems involved in generating reactive oxygen species.     

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C_m) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (π_o) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f_FM,i) increased with reference to the total fluorescence (f_FM,t) by 7.4 × 10⁻⁴ min⁻¹. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of π_o by 200 mosmol kg⁻¹ caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time f_FM,i/f_FM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of C_m and total fluorescence of a protoplast (f_FM,p) showed that an increase in π_o evoked a decrease in C_m but no change in f_FM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. Received: 4 May 1999 / Accepted: 19 August 1999     

Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3–6 h after the infection. The H₂O₂ production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H₂O₂ depleted the medium of H⁺ but the expected alkalinization of about one pH-unit did not occur. The H₂O₂ production by the plasma membrane oxidase must therefore be charge-compensated, likely via H⁺-channel activity. Received: 4 October 1997 / Accepted: 12 May 1998     

Tissue transglutaminase and the stress response

Summary. The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions. Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines. These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions.     

Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes

A cDNA encoding a novel inwardly rectifying potassium (K⁺ _in) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K⁺ _in channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K⁺-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than −50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channel's pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺ _in channels. The pharmacological inhibitor Cs⁺, applied externally, inhibited K⁺ _in currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC₅₀) of 21 μM and 17 μM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K⁺ into root hair cells. Comparison of homologous K⁺ _in rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships. Received: 3 August 1999 / Accepted: 2 November 1999     

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana

 Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO₂ fluxes in cell suspensions using mass spectrometry. Addition of H¹³CO₃ ⁻ to cell suspensions in the dark caused a transient increase in the CO₂ concentration in the medium far in excess of the equilibrium CO₂ concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO₂ efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO₂ by active transport. Photosynthetic O₂ evolution and the release CO₂ in the dark depend on HCO₃ ⁻ uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO₂ efflux in the dark, indicating that CO₂ efflux was dependent upon the intracellular dehydration of HCO₃ ⁻. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO₂ and HCO₃ ⁻ and thus causes a subsequent release of CO₂ to the external medium. Received: 20 September 1999 / Accepted: 25 October 1999     

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus

 The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A₁ (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions. Received: 19 February 1999 / Accepted: 14 August 1999     

In planta measurements of oxidative bursts elicited by avirulent and virulent bacterial pathogens suggests that H2O2 is insufficient to elicit cell death in tobacco

A rapid and localized programmed cell death – the hypersensitive response (HR) – is a widely utilized plant resistance mechanism against pathogens. Studies have implicated H₂O₂ generation as a key elicitory mechanism in the HR. The causal relationship between the kinetics of the in planta oxidative burst, the HR and certain defence gene expression was examined. H₂O₂ generation following challenge with avirulent strains of Pseudomonas syringae pv. (P. s. pv.) syringae occurred in two phases. The effects of ROS generation were investigated using the H₂O₂-responsive transgene AoPR10-GUS, the dually responsive (H₂O₂ and salicylic acid) PR1a-GUS as well as measures of cell death. Co-application of catalase with P. s. pv. syringae into tobacco leaf panels suppressed AoPR10- and PR1a-GUS expression and cell death. Conversely, varying H₂O₂ generation with glucose: glucose oxidase influenced both defence gene expression and cell death. AoPR10-GUS proved to be primarily responsive to apoplastic not intracellular oxidative stress, suggesting that the apoplasm was a distinctive source of oxidative signals. A biphasic oxidative burst was also observed with virulent P. s. pv. tabaci, which, although delayed compared to that observed during HR, persisted at equivalent levels for a longer period. Taking all these data together we suggest that either (1) additional factors to the apoplastic oxidative burst are required to explain the rapid kinetics of defence signalling and cell death associated with the HR or (2) P. s. pv. tabaci successfully suppresses the effects of H₂O₂ generation by an unknown mechanism.     

Oligocarrageenans and tissue-dependant oxidative burst in Solieria chordalis (Rhodophyceae, Gigartinales)

The release of hydrogen peroxide by thallus fragments of the rhodophycean Solieria chordalis (C. Agardh) J. Agardh has been documented both in the presence and in the absence of oligosaccharides. Within 1 h, ramuli were able to release large amounts of peroxide in the absence of any chemical stress. Among potential elicitors tested, only degree of polymerization 1 (DP1) and DP7‐8 oligo‐iota‐carrageenans stimulated defense mechanisms in both axes and ramuli as shown by the occurrence of an oxidative burst. Chopping of the tissues had no effect on the intensity of the burst, therefore suggesting that mainly cortical cell layers were involved in the process. After 5 min incubation, a dose of 125 μg mL^?1 of an oligomeric mixture containing a large proportion of DP1 units proved to be sufficient to obtain a maximal response. The intensity of the burst was significantly higher with isolated ramuli than with pieces of the axis, with outer peroxide accumulations reaching 200 nmol g^?1 fresh weight of treated tissue. Altogether, our results show that S. chordalis is able to react to a simulated pathogen attack by an oxidative burst and that the capacity to carry out an oxidative burst is stronger in ramuli than in axes.     

Suppression of an elicitor-induced oxidative burst reaction in Nicotiana tabacum and Medicago sativa cell cultures by corticrocin but not by mycorradicin

The biological activities of mycorradicin, the major component of the yellow pigment formed in maize and other grasses upon colonization by arbuscular mycorrhizal fungi, and corticrocin from the ectomycorrhizal fungus Piloderma croceum were analysed in cell cultures of tobacco (Nicotiana tabacum L.) and alfalfa (Medicago sativa L.). Tobacco and alfalfa suspension cell cultures react to elicitor treatment by alkalinization of the culture medium and generation of activated oxygen species, the so-called oxidative burst. In the present study, the addition of corticrocin suppressed the elicitor-induced oxidative burst reaction but not the alkalinization. The suppression of the oxidative burst by corticrocin was dose dependent. Mycorradicin in either its methylated or free form had no effect on the oxidative burst or the alkalinization. Accepted: 20 March 2001     

Protective effect of taurine on respiratory burst activity of polymorphonuclear leukocytes in endotoxemia

Summary. The aim of this study was to evaluate the effect of endotoxin on PMN leukocyte respiratory burst activity by measuring G6PD, NADPH oxidase and XO activities in guinea pig. In addition, the possible protective role of taurine against endotoxin-mediated PMN leukocyte function was examined. All experiments were performed with four groups (control, taurine, endotoxemia, taurine plus endotoxin) of ten guinea pigs. After the endotoxin was administrated (4 mg/kg) both G6PD and NADPH oxidase activities were significantly reduced compared with the control group. NADPH oxidase activity returned to the control value and G6PD activity also increased but it did not reach the control value. However when taurine was administrated (300 mg/kg) the activity of NADPH oxidase reached the control value; furthermore, G6PD activity also increased but it could not reach to the control value. When taurine was administrated alone, no effect on these enzymes was observed. Following the endotoxin administration, the activity of XO considerably increased. When taurine was administrated together with endotoxine and alone, this activity decreased compared to control value in both conditions. These results indicate that the O₂ ^•− formation in PMN leukocytes after the endotoxin administration is ensured by the catalysis of XO due to the inhibited NADPH oxidase activity. It was observed that taurine has considerable anti-inflammatory and antioxidant effects. However, conflicting results were obtained when taurine was administrated alone or together with an oxidant agent.     

Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: a 31P-nuclear magnetic resonance in-vivo study

The metabolic responses occurring in cucumber (Cucumis sativus L.) roots (a strategy-I plant) grown under iron-deficiency conditions were studied in-vivo using ³¹P-nuclear magnetic resonance spectroscopy. Iron starvation induced activation of metabolism leading to the consumption of stored carbohydrates to produce the NAD(P)H, ATP and phosphoenolpyruvate necessary to sustain the increased activity of the NAD(P)H:Fe³⁺-reductase, the H⁺-ATPase (EC 3.6.1.35) and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Activation of catabolic pathways was supported by the enhancement of glycolytic enzymes and concentrations of the metabolites glucose-6-phosphate and fructose-6-phosphate, and by enhancement of the respiration rate. Moreover, Fe-deficiency induced a slight increase in the cytoplasmic (pH_c) and vacuolar (pH_v) pHs as well as a dramatic decrease in the vacuolar phosphate (Pi) concentration. A comparison was done using fusicoccin (FC), a fungal toxin which stimulates proton extrusion. Changes in pH_c and pH_v were measured after addition of FC. Under these conditions, a dramatic alkalinization of the pH_v of −Fe roots was observed, as well as a concomitant Pi movement from the vacuole to the cytoplasm. These results showed that Fe starvation was indeed accompanied by the activation of metabolic processes useful for sustaining the typical responses occurring at the plasma-membrane level (i.e. increases in the NAD(P)H:Fe³⁺-reductase and H⁺-ATPase activities) as well as those involved in the homeostasis of pH_c. The decrease in vacuolar Pi levels induced by Fe-deficiency and FC and movement of Pi from the vacuole to the cytoplasm suggest a possible involvement of this compound in the cellular pH-stat system. Received: 30 July 1999 / Accepted: 11 November 1999     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Summary.  Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H⁺ efflux rate, 1.80 μM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H⁺ consumption for O₂ ^•− dismutation to H₂O₂. Also K⁺ influx was strongly depressed by MeJA, even transitorily reverting to K⁺ efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H₂O₂ accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O₂ uptake by roots gave similar results. These and other results for additions of H₂O₂ or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H₂O₂ being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN⁻- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O₂ ^•− and H₂O₂, O₂ uptake, and peroxidase activity by roots. Received July 12, 2002; accepted October 2, 2002; published online May 21, 2003 RID="*"     

Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore

Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plant's defense response. Received: 13 June 1999 / Accepted: 21 August 1999