Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E−. Plants infected by N. lolii exhibited higher production of phenolics relative to the E− plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, β-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P < 0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P < 0.05) and β-caryophyllene (P < 0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants’ protection.     

Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na⁺. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl₂, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The K_m for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.     

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: Accumulation and function of QB-nonreducing centers

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in F_m relative to F_o, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of Q_B-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of Q_B-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Dinitrosyliron complexes are the most abundant nitric oxide-derived cellular adduct: biological parameters of assembly and disappearance

It is well established that nitric oxide (^•NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all ^•NO-derived adducts (900 pmol/mg protein, or 45-90 μM). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous and exogenous sources of ^•NO in relation to iron and O₂ concentration. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of ^•NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic ^•NO concentrations (50-500 nM). Decreasing the O₂ concentration reduced the rate of enzymatic ^•NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t_1/2 = 80 min) and remained detectable long after the ^•NO source was removed (> 24 h). These results demonstrate that DNIC will be formed under all cellular settings of ^•NO production and that the contribution of DNIC to the multitude of observed effects of ^•NO must always be considered.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680 and the quinone acceptors QA to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q_A and Q_B, and the S₂ state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q_A⁻ reoxidation.     

Photosynthetic response of clusterbean chloroplasts to UV-B radiation: Energy imbalance and loss in redox homeostasis between QA and QB of photosystem II

The effects of ultraviolet-B (UV-B: 280-320 nm) radiation on the photosynthetic pigments, primary photochemical reactions of thylakoids and the rate of carbon assimilation (P_n) in the cotyledons of clusterbean (Cyamopsis tetragonoloba) seedlings have been examined. The radiation induces an imbalance between the energy absorbed through the photophysical process of photosystem (PS) II and the energy consumed for carbon assimilation. Decline in the primary photochemistry of PS II induced by UV-B in the background of relatively stable P_n, has been implicated in the creation of the energy imbalance_. The radiation induced damage of PS II hinders the flow of electron from Q_A to Q_B resulting in a loss in the redox homeostasis between the Q_A to Q_B leading to an accumulation of Q_A⁻. The accumulation of Q_A⁻ generates an excitation pressure that diminishes the PS II-mediated O₂ evolution, maximal photochemical potential (F_v/F_m) and PS II quantum yield (Φ_{PS II}). While UV-B radiation inactivates the carotenoid-mediated protective mechanisms, the accumulation of flavonoids seems to have a small role in protecting the photosynthetic apparatus from UV-B onslaught. The failure of protective mechanisms makes PS II further vulnerable to the radiation and facilitates the accumulation of malondialdehyde (MDA) indicating the involvement of reactive oxygen species (ROS) metabolism in UV-B-induced damage of photosynthetic apparatus of clusterbean cotyledons.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

Evolutionary relatedness of mackerels of the genus Scomber based on complete mitochondrial genomes: Strong support to the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as distinct species

Mackerels of the genus Scomber are commercially important species, but their taxonomic status is still controversial. Although previous phylogenetic data support the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as separate species, it is only based on the analysis of partial mitochondrial and nuclear DNA sequences. In an attempt to shed light on this relevant issue, we have determined the complete mitochondrial DNA sequence of S. colias, S. japonicus, and Scomber australasicus. The total length of the mitogenomes was 16,568 bp for S. colias and 16,570 bp for both S. japonicus and S. australasicus. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs, and 22 tRNAs) and organization similar to that observed in Scomber scombrus and most other vertebrates. The major noncoding region (control region) ranged between 865 and 866 bp in length and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Scomber species with regard to other scombrid fish. The major finding of this study is that S. colias and S. japonicus were significantly grouped in distinct lineages within Scomber cluster, which phylogenetically constitutes evidence that they may be considered as separate species. Additionally, molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.     

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: The qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700⁺ and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP⁺-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 μmol photons m⁻² s⁻¹ did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to ‘close’ all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q_A a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q_B-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.     

Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase

Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modified gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied under anaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry. On cyclic voltammograms nonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observed corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively. The immobilized BOD was differently oriented on the two electrodes and its catalysis of O₂-electroreduction was also massively different. On SPGE, where most of the enzyme was oriented with the T1 copper site proximal to the carbon with a quite slow ET process, well-pronounced DET-bioelectroreduction of O₂ was observed, starting already at > 700 mV vs. NHE. In contrast, on MPA-gold most of the enzyme was oriented with its T2/T3 copper cluster proximal to the metal. Indeed, there was little DET-based catalysis of O₂-electroreduction, even though the ET between the MPA-gold and the T2/T3 copper cluster of BOD was similar to that observed for the T1 site at SPGE. When BOD actively catalyzes the O₂-electroreduction, the redox potential of its T1 site is 690 mV vs. NHE and that of one of its T2/T3 copper centers is 390 mV vs. NHE. The redox potential of the T2/T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs. NHE. These values, combined with the observed biocatalytic behavior, strongly suggest an uphill intra-molecular electron transfer from the T1 site to the T2/T3 cluster during the catalytic turnover of the enzyme.     

Kinetic study of the plastoquinone pool availability correlated with H2O2 release in seawater and antioxidant responses in the red alga Kappaphycus alvarezii exposed to single or combined high light, chilling and chemical stresses

Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H₂O₂ into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H₂O₂ in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H₂O₂ release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H₂O₂ concentration in seawater (R = 0.673), total superoxide dismutase activity (R = 0.689), and particularly indexes of protein (R = 0.869) and lipid oxidation (R = 0.864), were moderately correlated. These data suggest that the release of H₂O₂ from plastids into seawater possibly impaired efficient and immediate responses of pivotal H₂O₂-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.     

Effects of precipitation on photosynthesis and water potential in Andropogon gerardii and Schizachyrium scoparium in a southern mixed grass prairie

In grassland ecosystems, spatial and temporal variability in precipitation is a key driver of species distributions and population dynamics. We experimentally manipulated precipitation to understand the physiological basis for differences in responses of species to water availability in a southern mixed grass prairie. We focused on the performance of two dominant C₄ grasses, Andropogon gerardii Vitman and Schizachyrium scoparium (Michx.) Nash, in treatments that received ambient rainfall, half of ambient rainfall (“drought” treatment), or approximately double ambient rainfall (“irrigated” treatment). Water potentials of S. scoparium were lower than A. gerardii, suggesting superior ability to adjust to water deficit in S. scoparium. Additionally, drought reduced photosynthesis to a greater extent in A. gerardii compared to S. scoparium. Leaf-level photosynthesis rates were similar in ambient and irrigated treatments, but were significantly lower in the drought treatment. Although stomatal conductance was reduced by drought, this was not limiting for photosynthesis. Leaf δ¹³C values were decreased by drought, caused by an increase in C_i/C_a. Chlorophyll fluorescence measures indicated light-harvesting rates were highest in irrigated treatments, and were lower in ambient and drought treatments. Moreover, drought resulted in a greater proportion of absorbed photon energy being lost via thermal pathways. Reductions in photosynthesis came as a result of non-stomatal limitations in the C₄ cycle. Our results provide mechanistic support for the hypothesis that S. scoparium is more drought tolerant than A. gerardii.