Alkalinization Of Medium Research Articles

Medium acidosis drives cardiac differentiation during mesendoderm cell fate specification from human pluripotent stem cells

Effective lineage-specific differentiation is essential to fulfilling the great potentials of human pluripotent stem cells (hPSCs). In this report, we investigate how modulation of medium pH and associated metabolic changes influence mesendoderm differentiation from hPSCs. We show that daily medium pH fluctuations are critical for the heterogeneity of cell fates in the absence of exogenous inducers. Acidic environment alone leads to cardiomyocyte generation without other signaling modulators. In contrast, medium alkalinization is inhibitory to cardiac fate even in the presence of classic cardiac inducers. We then demonstrate that acidic environment suppresses glycolysis to facilitate cardiac differentiation, while alkaline condition promotes glycolysis and diverts the differentiation toward other cell types. We further show that glycolysis inhibition or AMPK activation can rescue cardiac differentiation under alkalinization, and glycolysis inhibition alone can drive cardiac cell fate. This study highlights that pH changes remodel metabolic patterns and modulate signaling pathways to control cell fate.

A complete and sustained organic/inorganic reaction mechanism of Baeyer’s test

The Baeyer’s test for unsaturation has been used in Qualitative Organic Analysis for a long time. However, if an alkaline reagent is used, compounds having an active hydrogen (carbon acids) also give a positive test with potassium permanganate, detracting the test for unsaturation. The first steps of the reaction sequence have been described but the next stages are missing. These are very important because they involve the formation mode of the observed end product, brown manganese dioxide. In this communication, a complete and sustained reaction mechanism is provided for both reaction mediums, neutral and alkaline. It is in accordance with observed experimental facts. The missing steps are a redox reaction between hypomanganate and permanganate ions, and a second cyclic intermediate now formed with manganate ion. The instability of this intermediate leads directly to manganese dioxide and alkalinization of the neutral medium, as observed experimentally.

The chemistry of Anderson’s test for codeine

The Anderson test for codeine entails the interaction of codeine with concentrated nitric acid and heating to dryness. These drastic conditions give rise to a long series of reactions that is provided for the first time. The initial steps are salt formation, codeine nitrate, followed by nitration of the aromatic ring at the activated position, C-2. Then occurs a molecular rearrangement analogue to the transformation of morphine to apomorphine. This implies a series of reactions that include dehydration of the allylic alcohol, double bond migration, 1,3-chain migration, cyclohexadiene formation, ring opening of the dihydro-furane ring followed by formation of a dihydro-phenanthrene structure. The last stage involves methylamine evolution after two C─N fissions and alkalinization of the reaction medium in order to obtain the free amine.

A membrane protein of the rice pathogen Burkholderia glumae required for oxalic acid secretion and quorum sensing.

Bacterial panicle blight is caused by Burkholderia glumae and results in damage to rice crops worldwide. Virulence of B. glumae requires quorum sensing (QS)-dependent synthesis and export of toxoflavin, responsible for much of the damage to rice. The DedA family is a conserved membrane protein family found in all bacterial species. B. glumae possesses a member of the DedA family, named DbcA, which we previously showed is required for toxoflavin secretion and virulence in a rice model of infection. B. glumae secretes oxalic acid as a "common good" in a QS-dependent manner to combat toxic alkalinization of the growth medium during the stationary phase. Here, we show that B. glumae ΔdbcA fails to secrete oxalic acid, leading to alkaline toxicity and sensitivity to divalent cations, suggesting a role for DbcA in oxalic acid secretion. B. glumae ΔdbcA accumulated less acyl-homoserine lactone (AHL) QS signalling molecules as the bacteria entered the stationary phase, probably due to nonenzymatic inactivation of AHL at alkaline pH. Transcription of toxoflavin and oxalic acid operons was down-regulated in ΔdbcA. Alteration of the proton motive force with sodium bicarbonate also reduced oxalic acid secretion and expression of QS-dependent genes. Overall, the data show that DbcA is required for oxalic acid secretion in a proton motive force-dependent manner, which is critical for QS of B. glumae. Moreover, this study supports the idea that sodium bicarbonate may serve as a chemical for treatment of bacterial panicle blight.

Fusarium oxysporum Casein Kinase 1, a Negative Regulator of the Plasma Membrane H+-ATPase Pma1, Is Required for Development and Pathogenicity.

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum.

Polyhydroxybutyrate production in one-stage by purple phototrophic bacteria: Influence of alkaline pH, ethanol, and C/N ratios

This work aimed to evaluate the effect of alkaline pH, nutrient limitation, nitrogen source, C/N ratio, and ethanol addition on polyhydroxybutyrate (PHB) production by a photoheterotrophic mixed culture enriched in Rhodopseudomonas palustris. The results demonstrated the natural alkalinization of the culture medium was enough to induce PHB accumulation in the mixed culture. The combination of alkaline pH, nitrogen, sulfur, or phosphate deficiency negatively affected polymer production. The incorporation of glutamate for biomass synthesis was better than that of ammonium, which had a positive effect on PHB production. Interestingly, ethanol addition improved the PHB accumulation by 25 %, but without consumption. PHB production improved at low C/N ratios, where 27 ± 0.9% of the carbon source was transformed into biopolymers. The evaluated factors allowed the biomass PHB content to increase by 60%, reaching PHB production of 219 ± 5 mg/L. The PHB production by the photoheterotrophic mixed culture is associated with growth, allowing the PHB production in one stage. That has significant advantages in technical-practical terms, such as reducing the time of the process, dispensing with the separation of biomass from the feast phase to the famine phase, and requiring a single reactor.

Aggregation of morpholinium surfactants with amino alcohols as additives: a close look

Mono-, di- and triethanolamine in combination with methylmorpholinium and hydroxyethylmorpholinium surfactants were investigated for their aggregation and solubilizing properties. A co-operative behavior of the solubilization by mixed surfactant–ethanolamine systems is described. Ethanolamines strongly affect pH and lead to Orange OT phenolic group deprotonation and subsequent increase in aqueous/micellar solubility. The morpholinium surfactant micelles reduce the pK a of the Orange OT phenolic group, enabling its deprotonation at the earlier stages of medium alkalinization. The obtained surfactant–ethanolamine mixtures can solubilize very large amounts of hydrophobic dye, which can then be triggered to precipitate through acidification.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Relationships between Plant Defense Inducer Activities and Molecular Structure of Gallomolecules.

Plant defense inducers (PDIs) are booming and attractive protection agents designed to immunostimulate the plant to reduce subsequent pathogen colonization. The structure-PDI activity relationships of four flavan-3-ols: Epicatechin (EC), Epigallocatechin (EGC), Epicatechin gallate (ECG), Epigallocatechin gallate (EGCG) and Gallotannic acid (GTA) were investigated in both whole plant and suspension cell systems. ECG, EGCG, and GTA displayed elicitor activities. Their infiltration into tobacco leaves induced hypersensitive reaction-like lesions with topical scopoletin and PR-target transcript accumulations. On the contrary, EC and EGC infiltrations fail to trigger the biochemical changes in tobacco tissues. The tobacco BY-2 cells challenged with ECG, EGCG, or GTA led to alkalinization of the BY-2 extracellular medium while EC and EGC did not trigger any pH variation. This work provides evidence that the esterified gallate pattern is as an essential flavonoid entity to induce plant defense reactions in tobacco. The phytoprotective properties of the esterified gallate-free EC and the esterified gallate-rich GTA were evaluated on the tobacco/Phytophthora parasitica var. nicotianae (Ppn) pathosystem. Tobacco treatment with EC did not induce significant protection against Ppn compared to GTA which shows antimicrobial properties on Ppn and decreases the infection on GTA-infiltrated and -sprayed wild-type leaves. GTA protection was impaired in the transgenic NahG tobacco plants, suggesting that protection was mediated by salicylic acid.

Volatile-Mediated Inhibitory Activity of Rhizobacteria as a Result of Multiple Factors Interaction: The Case of Lysobacter capsici AZ78.

Plant beneficial rhizobacteria may antagonize soilborne plant pathogens by producing a vast array of volatile organic compounds (VOCs). The production of these compounds depends on the medium composition used for bacterial cell growth. Accordingly, Lysobacter capsici AZ78 (AZ78) grown on a protein-rich medium was previously found to emit volatile pyrazines with toxic activity against soilborne phypathogenic fungi and oomycetes. However, the discrepancy between the quantity of pyrazines in the gaseous phase and the minimum quantity needed to achieve inhibition of plant pathogens observed, lead us to further investigate the volatile-mediated inhibitory activity of AZ78. Here, we show that, besides VOCs, AZ78 cells produce ammonia in increased amounts when a protein-rich medium is used for bacterial growth. The production of this volatile compound caused the alkalinization of the physically separated culture medium where Rhizoctonia solani was inoculated subsequently. Results achieved in this work clearly demonstrate that VOC, ammonia and the growth medium alkalinization contribute to the overall inhibitory activity of AZ78 against R. solani. Thus, our findings suggest that the volatile-mediated inhibitory activity of rhizobacteria in protein-rich substrates can be regarded as a result of multiple factors interaction, rather than exclusively VOCs production.

MAMP-triggered Medium Alkalinization of Plant Cell Cultures.

Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.

On the Colloidal Stability of Nitrogen-Rich Carbon Nanodots Aqueous Dispersions

The present survey reports on the colloidal stability of aqueous dispersions of nitrogen-rich carbon nanodots (N-CDs). The N-CDs were synthesized by thermally induced decomposition of organic precursors and present an inner core constituted of a β − C 3 N 4 crystalline structure surrounded by a surface shell containing a variety of polar functional groups. N-CDs size and structure were checked by combined analysis of XRD (X-ray Diffraction) and TEM (Transmission Electron Microscopy) measurements. FTIR (Fourier-Transform Infrared Spectroscopy) experiments revealed the presence of carboxyl and amide groups on N-CDs surface. Towards a better understanding of the relation between colloidal stability and surface charge development, zetametry experiments were applied in N-CDs dispersions at different pHs and constant ionic strength. The increase of the absolute values of zeta potential with the alkalinization of the dispersion medium is consistent with the deprotonation of carboxyl groups on N-CDs surface, which agrees with the macroscopic visual observations of long-term colloidal stability at pH 12. The saturation value of N-CDs surface charge density was evaluated by means of potentiometric-conductometric titrations. The difference between carboxyl-related surface charge and the one determined by zeta potential measurements point to the presence of oxidized nitrogen functionalities onto the N-CDs surface in addition to carboxyl groups. These novel results shed light on the electrostatic repulsion mechanism that allows for the remarkable colloidal stability of N-CDs dispersions.

The effect of lead pollution on nutrient solution pH and concomitant changes in plant physiology of two contrasting Solanum melongena L. cultivars.

Lead (Pb) is highly toxic to plants because it severely affects physiological processes by altering nutrient solution pH. The current study elucidated Pb-induced changes in nutrient solution pH and its effect on physiology of two Solanum melongena L. cultivars (cv. Chuttu and cv. VRIB-13). Plants were grown in black plastic containers having 0, 15, 20, and 25 mg L-1 PbCl2 in nutrient solutions with starting pH of 6.0. pH changes by roots of S. melongena were continuously monitored for 8 days, and harvested plants were analyzed for physiological and biochemical attributes. Time scale studies revealed that cv. Chuttu and cv. VRIB-13 responded to Pb stress by causing acidification and alkalinization of growth medium during the first 48 h, respectively. Both cultivars increased nutrient solution pH, and maximum pH rise of 1.21 units was culminated by cv. VRIB-13 at 15 mg L-1 Pb and 0.8 units by cv. Chuttu at 25 mg L-1 Pb treatment during the 8-day period. Plant biomass, photosynthetic pigments, ascorbic acid, total amino acid, and total protein contents were significantly reduced by Pb stress predominantly in cv. Chuttu than cv. VRIB-13. Interestingly, chlorophyll contents of cv. VRIB-13 increased with increasing Pb levels. Pb contents of roots and shoots of both cultivars increased with applied Pb levels while nutrient (Ca, Mg, K, and Fe) contents decreased predominately in cv. Chuttu. Negative correlations were identified among Pb contents of eggplant roots and shoots and plant biomasses, leaf area, and free anthocyanin. Taken together, growth medium alkalinization, lower root to shoot Pb translocation, and optimum balance of nutrients (Mg and Fe) conferred growth enhancement, ultimately making cv. VRIB-13 auspicious for tolerating Pb toxicity as compared with cv. Chuttu. The research outcomes are important for devising metallicolous plant-associated strategies based on plant pH modulation response and associated metal uptake to remediate Pb-polluted soil.

Behavioral Characteristics of Mice with Knockout of the IRR Alkali Sensor Gene

The tyrosine kinase receptor IRR (insulin receptor-related receptor) can be activated on alkalinization of the cell medium. IRR is detected in organs in contact with fluids with extreme pH values, as well as in certain central nervous system cells, where its role is unclear. We report here our analysis of the behavioral characteristics of mice with knockout of the IRR gene in a series of ethological tests. The control group included animals with the IRR gene from the same litters as knockout mice. In the social interest test, knockout animals showed a reduction in the number of social contacts. The forced swimming test revealed no statistically significant differences in the duration of immobility, though there were more animals showing longer periods of immobility among knockout mice. In the resident-intruder test, wild-type mice showed typical aggression levels, while seven of 16 knockout animals remained inert and, conversely, were attacked by the “stranger.” These data provide evidence that deletion of the IRR gene leads to impairment of the aggressive-defensive behavior typical of the parental strain.

Secreted proteins produced by fungi associated with Botryosphaeria dieback trigger distinct defense responses in Vitis vinifera and Vitis rupestris cells.

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.

Ethyl Gallate Displays Elicitor Activities in Tobacco Plants.

Alkyl gallates showed elicitor activities on tobacco in both whole plants and cell suspensions. Methyl gallate (MG), ethyl gallate (EG), and propyl gallate (PG) infiltration into tobacco leaves induced hypersensitive reaction-like lesions and topical production of autofluorescent compounds revealed under UV light. When sprayed on tobacco plants at 5 mM, EG promoted upregulation of defense-related genes such as the antimicrobial PR1, β-1,3-glucanase PR2, Chitinase PR3, and osmotin PR5 target genes. Tobacco BY-2 cells challenged with EG underwent cell death in 48 h, which was significantly reduced in the presence of the protease inhibitor aprotinin. The three alkyl gallates all caused alkalinization of the BY-2 extracellular medium, whereas gallic acid did not trigger any pH variation. Using EGTA or LaCl3, we showed that Ca2+ mobilization occurred in BY-2 cells elicited with EG. Overall, our findings are the first evidence of alkyl gallate elicitor properties with early perception events on the plasma membrane, potential hypersensitive reactions, and PR-related downstream defense responses in tobacco.

Pathways for external alkalinization in intact and in microwounded Chara cells are differentially sensitive to wortmannin

ABSTRACTProton flows across the plant cell membranes play a major role in electrogenesis and regulation of photosynthesis and ion balance. The profiles of external pH along the illuminated internodal cells of characean algae consist of alternating high- and low-pH zones that are spatially coordinated with the distribution of photosynthetic activity of chloroplasts underlying these zones. The results based on confocal laser scanning fluorescence microscopy, pH microsensors, and pulse-amplitude-modulated chlorophyll microfluorometry revealed that the coordination of H+ transport and photosynthesis is disrupted by the 2 different environmental cues (low light and wounding) and by a chemical, wortmannin interfering with the inositol phospholipid metabolism. On the one hand, the transition from moderate to low irradiance diminished the peaks in the profiles of photosystem II (PSII) quantum efficiency but did not remove the pH bands. On the other hand, the microwounding of the internode with a glass micropipette, impacting primarily the cell wall, resulted in a rapid local alkalinization of the external medium (by 2–2.5 pH units) near the cell surface, thus mimicking the appearance of natural pH bands. Despite their seeming similarity, the alkaline bands of intact cells were eliminated by wortmannin, whereas the wound-induced alkalinization was insensitive to this drug. Furthermore, the attenuation of natural pH bands in wortmannin-treated cells was accompanied by the enhancement in spatial heterogeneity of PSII efficiency and electron transport rates, which indicates the complexity of chloroplast–plasma membrane interactions. The results suggest that the light- and wound-induced alkaline areas on the cell surface are associated with different ion-transport systems.

Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

The Impact of Protein Binding on Antibacterial Activities of Antibiotics is more than Predicted by Considering its Numerical Value Alone: Impact of Preparative and Incubation Methods on Different Pharmacodynamic Endpoints of ß-Lactams, Macrolides, or Fluoroquinolones Against Gram-positive and Gram-negative Bacteria-Part I

Objectives: Protein binding decreases antibacterial activities as the free fraction only crosses membranes thus reaching intracellular targets. However, serum components may also increase antibacterial activities. Therefore, the effect of serum proteins on activities of ß-lactams, macrolides, and fluoroquinolones was examined. Several preparation- and cultivation-conditions were examined as some preparative methods like freeze-thawing of sera may cause artifacts. Furthermore, previous studies have indicated that data may vary depending on the endpoints studied. Therefore, the aim of this study was to avoid an impact of methodological factors on the data generated and to analyse the activities of the study drugs by examining different static- or dynamic endpoints. Methods: Bacteria were grown in Brain Heart Infusion Broth (BHI), plus 50% of either fresh inactivated, pH 7.2, or fresh inactivated- , pH 7.2 or 8.2, frozen inactivated-serum, pH 8.2 as compared to BHI without any supplementations, pH 7.2 or 8.2, and BHI plus 45 g/L albumin. MICs of faropenem, amoxicillin, clarithromycin, azithromycin, moxifloxacin, and levofloxacin were determined and kill-kinetics were recorded following exposure to constant or fluctuating drug concentrations simulating serum pharmacokinetics of the agents. Kill-rates and areas under the bacterial kill curves were calculated. Results: Albumin and inactive serum increased MICs and reduced kill-rates of the agents studied in conformity with their protein binding, whereas active serum increased the activities of the agents. MICs and kill-rates did not change in parallel. The impact of protein binding in decreasing order was: MICs>kill-rates in time-kill experiments>kill-rates in kinetic-simulations. Macrolides and fluoroquinolones but not ß-lactams were more active at an alkaline- than neutral pH. Use of frozen sera caused alkalinization of media, thus generating artifacts. Conclusions: The impact of serum proteins on antibacterial activities is strongly dependent from three factors: the methods applied to prepare the serum pool, the incubation conditions, and the enpoints studied.

The defence elicitor AsES causes a rapid and transient membrane depolarization, a triphasic oxidative burst and the accumulation of nitric oxide

The newly characterized elicitor AsES obtained from Acremonium strictum induces a strong defence response in strawberry plants and confers plants resistance against the fungal pathogen Colletotricum acutatum the casual agent of anthracnose disease. Previous studies showed that AsES causes the accumulation of reactive oxygen species (ROS) that peaked 4 h post treatment (hpt), but due to the experimental approach used it was not clear whether the accumulation of ROS observed was intracellular or extracellular or took place as a single peak. By using a different experimental setup, a more complex early events associated to the activation of the innate immunity were observed. In this paper we report that strawberry plant cells treated with AsES exhibits a triphasic production of H2O2 and a rapid intracellular accumulation of NO. The first phase consists in a progressive extracellular accumulation of H2O2 that starts immediately after the treatment with AsES and is preceded by a rapid and transient cell membrane depolarization. During this phase takes place also a rapid intracellular accumulation of NO. Microscopic observations of mesophyll cells treated with AsES reveals that NO accumulates at the chloroplast. After the first extracellular H2O2 production phase, two intracellular H2O2 accumulation events occur, the first 2 hpt, and the second 7 hpt. Cells treated with AsES also show a transient increase of ion leakage, and a progressive alkalinization of the extracellular medium.