Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH₄⁺-N per gram of soil) and high (200 μg NH₄⁺-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K_Mox values were around 4-5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK_Mox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K_Mox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K_Mox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K_Mox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

Spatial distribution of ammonia-oxidizing bacteria and archaea across a 44-hectare farm related to ecosystem functioning

Characterization of spatial patterns of functional microbial communities could facilitate the understanding of the relationships between the ecology of microbial communities, the biogeochemical processes they perform and the corresponding ecosystem functions. Because of the important role the ammonia-oxidizing bacteria (AOB) and archaea (AOA) have in nitrogen cycling and nitrate leaching, we explored the spatial distribution of their activity, abundance and community composition across a 44-ha large farm divided into an organic and an integrated farming system. The spatial patterns were mapped by geostatistical modeling and correlations to soil properties and ecosystem functioning in terms of nitrate leaching were determined. All measured community components for both AOB and AOA exhibited spatial patterns at the hectare scale. The patchy patterns of community structures did not reflect the farming systems, but the AOB community was weakly related to differences in soil pH and moisture, whereas the AOA community to differences in soil pH and clay content. Soil properties related differently to the size of the communities, with soil organic carbon and total nitrogen correlating positively to AOB abundance, while clay content and pH showed a negative correlation to AOA abundance. Contrasting spatial patterns were observed for the abundance distributions of the two groups indicating that the AOB and AOA may occupy different niches in agro-ecosystems. In addition, the two communities correlated differently to community and ecosystem functions. Our results suggest that the AOA, not the AOB, were contributing to nitrate leaching at the site by providing substrate for the nitrite oxidizers.     

内蒙古不同类型草地土壤氮矿化及其温度敏感性

土壤氮矿化(Nitrogen mineralization)是土壤氮循环的重要环节,对土壤氮素供应以及植物生产力的维持具有十分重要的意义。沿中国东北草地样带(Northeastern China Transect, NECT)分别在典型草地、过渡草地及荒漠草地设置了3个实验样地,利用不同温度(5、10、15、20 ℃和25 ℃)和不同水分(30%、60%和90%土壤饱和含水量,Saturated soil moisture, SSM)的室内培养途径,探讨了不同类型草地的土壤氮矿化速率、土壤氮矿化的温度敏感性(Q₁₀)及其主要影响因素。实验结果表明:从典型草地至荒漠草地,土壤全碳、全氮、全磷、微生物生物量碳氮含量均表现为逐渐下降的趋势;类似地,土壤净氮矿化速率、硝化速率也逐渐降低。在20 ℃和60% SSM时,土壤净氮矿化速率表现为典型草地 (0.715 mg N kg^-1 d^-1) > 过渡草地 (0.507 mg N kg^-1 d^-1) > 荒漠草地 (0.134 mg N kg^-1 d^-1);相反,温度敏感性却逐渐升高,温度敏感性与基质质量指数呈负相关。草地类型和水分对于土壤净氮矿化速率、硝化速率具有显著影响,且二者间具有显著的交互效应。包含温度和水分的双因素模型可很好地拟合土壤氮矿化速率的变化趋势(P < 0.0001),二者可共同解释土壤硝化速率92%-96%的变异。土壤氮矿化沿着草地演替呈现出很好的空间格局、并与温度和水分具有密切关系,为解释内蒙古草地空间分布格局提供了理论基础。     

An information-theoretic approach to evaluating the size and temperature dependence of metabolic rate

The effects of body mass and temperature on metabolic rate (MR) are among the most widely examined physiological relationships. Recently, these relationships have been incorporated into the metabolic theory of ecology (MTE) that links the ecology of populations, communities and ecosystems to the MR of individual organisms. The fundamental equation of MTE derives the relation between mass and MR using first principles and predicts the temperature dependence of MR based on biochemical kinetics. It is a deliberately simple, zeroth-order approximation that represents a baseline against which variation in real biological systems can be examined. In the present study, we evaluate the fundamental equation of MTE against other more parameter-rich models for MR using an information-theoretic approach to penalize the inclusion of additional parameters. Using a comparative database of MR measurements for 1359 species, from 11 groups ranging from prokaryotes to mammals, and spanning 16 orders of magnitude in mass and a 59°C range in body temperature, we show that differences between taxa in the mass and temperature dependence of MR are sufficiently large as to be retained in the best model for MR despite the requirement for estimation of 22 more parameters than the fundamental equation of MTE.     

Enzymatic oxidation of manganese ions catalysed by laccase

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.     

Acetate oxidation by syntrophic association between Geobacter sulfurreducens and a hydrogen-utilizing exoelectrogen

Anodic microbial communities in acetate-fed microbial fuel cells (MFCs) were analyzed using stable-isotope probing of 16S rRNA genes followed by denaturing gradient gel electrophoresis. The results revealed that Geobacter sulfurreducens and Hydrogenophaga sp. predominated in the anodic biofilm. Although the predominance of Geobacter sp. as acetoclastic exoelectrogens in acetate-fed MFC systems has been often reported, the ecophysiological role of Hydrogenophaga sp. is unknown. Therefore, we isolated and characterized a bacterium closely related to Hydrogenophaga sp. (designated strain AR20). The newly isolated strain AR20 could use molecular hydrogen (H₂), but not acetate, with carbon electrode as the electron acceptor, indicating that the strain AR20 was a hydrogenotrophic exoelectrogen. This evidence raises a hypothesis that acetate was oxidized by G. sulfurreducens in syntrophic cooperation with the strain AR20 as a hydrogen-consuming partner in the acetate-fed MFC. To prove this hypothesis, G. sulfurreducens strain PCA was cocultivated with the strain AR20 in the acetate-fed MFC without any dissolved electron acceptors. In the coculture MFC of G. sulfurreducens and strain AR20, current generation and acetate degradation were the highest, and the growth of strain AR20 was observed. No current generation, acetate degradation and cell growth occurred in the strain AR20 pure culture MFC. These results show for the first time that G. sulfurreducens can oxidize acetate in syntrophic cooperation with the isolated Hydrogenophaga sp. strain AR20, with electrode as the electron acceptor.     

Molecular,Physiological and Phenotypic Characterization of Paracoccus denitrificans ATCC 19367 Mutant Strain P-87 Producing Improved Coenzyme Q10

Coenzyme Q₁₀ (CoQ₁₀) is a blockbuster nutraceutical molecule which is often used as an oral supplement in the supportive therapy for cardiovascular diseases, cancer and neurodegenerative diseases. It is commercially produced by fermentation process, hence constructing the high yielding CoQ₁₀ producing strains is a pre-requisite for cost effective production. Paracoccus denitrificans ATCC 19367, a biochemically versatile organism was selected to carry out the studies on CoQ₁₀ yield improvement. The wild type strain was subjected to iterative rounds of mutagenesis using gamma rays and NTG, followed by selection on various inhibitors like CoQ₁₀ structural analogues and antibiotics. The screening of mutants were carried out using cane molasses based optimized medium with feeding strategies at shake flask level. In the course of study, the mutant P-87 having marked resistance to gentamicin showed 1.25-fold improvements in specific CoQ₁₀ content which was highest among all tested mutant strains. P-87 was phenotypically differentiated from the wild type strain on the basis of carbohydrate assimilation and FAME profile. Molecular differentiation technique based on AFLP profile showed intra specific polymorphism between wild type strain and P-87. This study demonstrated the beneficial outcome of induced mutations leading to gentamicin resistance for improvement of CoQ₁₀ production in P. denitrificans mutant strain P-87. To investigate the cause of gentamicin resistance, rpIF gene from P-87 and wild type was sequenced. No mutations were detected on the rpIF partial sequence of P-87; hence gentamicin resistance in P-87 could not be conferred with rpIF gene. However, detecting the mutations responsible for gentamicin resistance in P-87 and correlating its role in CoQ₁₀ overproduction is essential. Although only 1.25-fold improvement in specific CoQ₁₀ content was achieved through mutant P-87, this mutant showed very interesting characteristic, differentiating it from its wild type parent strain P. denitrificans ATCC 19367, which are presented in this paper.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0506-4) contains supplementary material, which is available to authorized users.     

放牧对若尔盖高寒草甸土壤氮矿化及其温度敏感性的影响

过度放牧和气候暖干化是若尔盖高寒草甸面临的严峻生态问题,它对土壤氮矿化过程的影响将在某种程度上决定高寒草甸生态系统的氮素利用状况。基于野外放牧实验,(禁牧(CK)、轻度放牧(L)、中度放牧(M)、重度放牧(H)),通过室内培养方法(5、10、15、20、25℃),探讨了不同放牧强度对若尔盖高寒草甸土壤氮矿化及其温度敏感性的影响。实验结果表明:放牧增加了土壤硝化速率和净氮矿化速率,其整体趋势为LMHCK。培养温度较低时(5—10℃),温度升高对硝化和净氮矿化速率无显著影响,而培养温度较高时(15—25℃),温度升高显著增加了硝化和净氮矿化速率。土壤硝态氮和无机氮积累量随培养时间增长显著增加,而铵态氮无显著增长。此外,放牧显著提高了土壤氮矿化的温度敏感性,重度放牧样地的土壤具有最高的氮矿化温度敏感性(Q10=2.72)。上述结果预示着在未来温度升高情景下,重度放牧将使土壤氮矿化速率的增速更快,短期内将通过提高氮的可利用性促进植物生长,但长远而言可能加速土壤氮素流失,从而对植物生长和高寒草甸生态系统结构与功能造成负反馈效应。     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q_a has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Q_a⁻ is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q_o of the cytochrome bc₁ complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q_a⁻ oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc₁ complex, which allows a fast transfer of quinone formed at the level of cyt bc₁ complex to the RCs. In agreement with this model, the fast phase of Q_a⁻ reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc₁. The PufX-deleted mutant displays only the slowest phase of Q_a⁻ oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc₁ and RCs.     

Characterization of active site residues of nitroalkane oxidase

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ∼5-fold and decreases in the rate constant for product release of ∼2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.     

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia,phylum Chloroflexi

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated ‘DEH-J10'' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO₂ or oxidation of organics completely to CO₂. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.     

Heritability and quantitative genetic divergence of serotiny,a fire-persistence plant trait

Background and Aims

Although it is well known that fire acts as a selective pressure shaping plant phenotypes, there are no quantitative estimates of the heritability of any trait related to plant persistence under recurrent fires, such as serotiny. In this study, the heritability of serotiny in Pinus halepensis is calculated, and an evaluation is made as to whether fire has left a selection signature on the level of serotiny among populations by comparing the genetic divergence of serotiny with the expected divergence of neutral molecular markers (Q_ST–F_ST comparison).

Methods

A common garden of P. halepensis was used, located in inland Spain and composed of 145 open-pollinated families from 29 provenances covering the entire natural range of P. halepensis in the Iberian Peninsula and Balearic Islands. Narrow-sense heritability (h²) and quantitative genetic differentiation among populations for serotiny (Q_ST) were estimated by means of an ‘animal model’ fitted by Bayesian inference. In order to determine whether genetic differentiation for serotiny is the result of differential natural selection, Q_ST estimates for serotiny were compared with F_ST estimates obtained from allozyme data. Finally, a test was made of whether levels of serotiny in the different provenances were related to different fire regimes, using summer rainfall as a proxy for fire regime in each provenance.

Key Results

Serotiny showed a significant narrow-sense heritability (h²) of 0·20 (credible interval 0·09–0·40). Quantitative genetic differentiation among provenances for serotiny (Q_ST = 0·44) was significantly higher than expected under a neutral process (F_ST = 0·12), suggesting adaptive differentiation. A significant negative relationship was found between the serotiny level of trees in the common garden and summer rainfall of their provenance sites.

Conclusions

Serotiny is a heritable trait in P. halepensis, and selection acts on it, giving rise to contrasting serotiny levels among populations depending on the fire regime, and supporting the role of fire in generating genetic divergence for adaptive traits.     

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10⁴ M^− 1 s^− 1, 30 M^− 1 s^− 1, 3.4 × 10³ M^− 1 s^− 1, and 8.6 × 10³ M^− 1 s^− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.     

Morphological and Molecular Differentiation of Sporidiobolus johnsonii ATCC 20490 and Its Coenzyme Q10 Overproducing Mutant Strain UF16

Coenzyme Q₁₀ (CoQ₁₀) is an industrially important molecule having nutraceutical and cosmeceutical applications. CoQ₁₀ is mainly produced by microbial fermentation and the process demands the use of strains with high productivity and yields of CoQ₁₀. During strain improvement program consisting of sequential induced mutagenesis, rational selection and screening process, a mutant strain UF16 was generated from Sporidiobolus johnsonii ATCC 20490 with 2.3-fold improvements in CoQ₁₀ content. EMS and UV rays were used as mutagenic agents for generating UF16 and it was rationally selected based on atorvastatin resistance as well as survival at free radicals exposure. We investigated the genotypic and phenotypic changes in UF16 in order to differentiate it from wild type strain. Morphologically it was distinct due to reduced pigmentation of colony, reduced cell size and significant reduction in mycelial growth forms with abundance of yeast forms. At molecular level, UF16 was differentiated based on PCR fingerprinting method of RAPD as well as large and small-subunit rRNA gene sequences. Rapid molecular technique of RAPD analysis using six primers showed 34 % polymorphic fragments with mean genetic distance of 0.235. The partial sequences of rRNA-gene revealed few mutation sites on nucleotide base pairs. However, the mutations detected on rRNA gene of UF16 were less than 1 % of total base pairs and its sequence showed 99 % homology with the wild type strain. These mutations in UF16 could not be linked to phenotypic or genotypic changes on CoQ₁₀ biosynthetic pathway that resulted in improved yield. Hence, investigating the mutations responsible for deregulation of CoQ₁₀ pathway is essential to understand the cause of overproduction in UF16. Phylogenetic analysis based on RAPD bands and rRNA gene sequences coupled with morphological variations, exhibited the novelty of mutant UF16 having potential for improved CoQ₁₀ production.     

植被类型变化对长白山森林土壤碳矿化及其温度敏感性的影响

土壤有机质是陆地生态系统最大的碳库,土壤有机质分解速率及其温度敏感性对生态系统碳循环及其碳汇功能具有重要影响。为揭示植被类型变化对森林土壤有机质分解的影响,以长白山针阔混交林的原生林和次生林为研究对象,分别将土壤在不同水分(30%、60%和90%土壤饱和含水量(SSM))和不同温度(5、10、15、20、25和30 ℃)下培养,在为期56 d的培养期内分9次测定土壤碳矿化速率。实验结果表明:植被类型、培养温度和水分对土壤碳矿化速率具有显著影响,且三者间存在显著的交互效应(P < 0.001)。次生林土壤碳矿化累积量显著高于原生林(P < 0.05),在90% SSM和温度30 ℃培养状况下分别为346.41 μgC/g和241.01 μgC/g。包含温度和水分的双因素模型可很好地拟合土壤碳矿化速率的变化,温度和水分可共同解释土壤碳矿化速率的82.7%-95.9%变异。次生林土壤碳矿化温度敏感性(Q₁₀)显著高于原生林;水分对温度敏感性的影响较复杂,次生林在60% SSM最高,而原生林在90% SSM最高。总之,原生林遭砍伐后将会加速土壤有机质的分解,从而降低土壤有机质含量;另外,根据Q₁₀值可以预测次生林土壤有机质的分解速率对全球变暖反映更明显。     

毛白杨树干呼吸及其温度敏感性季节变化特征

树干呼吸(E_s)是森林生态系统碳循环过程的重要组成部分,深入理解树干呼吸过程对未来气候变暖的响应及反馈机制有助于更加精确地估算森林生态系统碳储量。为揭示毛白杨树干呼吸及其温度敏感性的昼夜变化和季节动态规律,利用Li-Cor6400便携式光合作用测定系统及其配套使用的土壤呼吸测量气室(LI-6400-09)对冀南平原区毛白杨的树干呼吸和树干温度实施为期1年的连续监测。结果表明:(1)在生长季,毛白杨树干呼吸与树干温度之间在晚上呈现正相关的关系(R~2=0.88);相反,两者在白天为负相关的关系(R~2=0.96)。(2)整个观测期内,毛白杨树干呼吸和树干温度均呈现"钟形"的变化曲线,树干呼吸与树干温度之间存在着较好的指数函数关系(R~2=0.93),且树干呼吸的温度敏感性系数(Q_(10))为2.62;不同季节毛白杨树干呼吸的Q_(10)存在差异,生长季的Q_(10)(1.95)明显低于非生长季(3.00),表明生长呼吸和维持呼吸对温度的响应也并不相同。(3)温度矫正后的毛白杨树干呼吸(R_(15))在昼夜和季节尺度上均存在明显的变异,即夜晚的R_(15)显著高于白天(P0.01),生长季的R_(15)明显高于非生长季(P0.05);树干可溶性糖含量与生长季的R_(15)存在较好的相关性(R~2=0.52),而非生长季的R_(15)却主要受到树干淀粉含量的影响。研究结果表明,在生长季,毛白杨树干呼吸的在日变化主要受到温度的影响,而在季节尺度上Q_(10)的变异则与树干呼吸中维持呼吸所占比例及树干中非结构性碳水化合物(可溶性糖和淀粉)的含量及类型紧密相关。     

Anoxic depolarization of hippocampal astrocytes: Possible modulation by P2X7 receptors

Current responses from CA1 neurons and stratum oriens astrocytes were recorded from hippocampal brain slices by means of the whole-cell patch-clamp technique. Anoxic depolarization (AD) was induced by an oxygen/glucose-deprived (OGD) medium also containing sodium iodoacetate and antimycin, in order to block glycolysis and oxidative phosphorylation, respectively. Anoxic depolarization has been reported to be due to the sudden increase of the extracellular K⁺ concentration and the accompanying explosive rise in glutamate concentration. We asked ourselves whether the release of ATP activating P2X7 receptors is also involved in the AD. Although, the AD was evoked in absolute synchrony in neurons and astrocytes, and the NMDA receptor antagonistic AP-5 depressed these responses, neither the non-selective P2 receptor antagonist PPADS, nor the highly selective P2X7 receptor antagonist A438079 interfered with the AD or its delay time in neurons/astrocytes after inducing chemical hypoxia. However, A438079, but not PPADS increased in astrocytes the slow inward current observed in a hypoxic medium. It is concluded that ATP co-released with glutamate by hypoxic stimulation has only a minor function in the present brain slice system.     

The impact of cold on photosynthesis in genotypes of Coffea spp.--photosystem sensitivity, photoprotective mechanisms and gene expression

Environmental constraints disturb plant metabolism and are often associated with photosynthetic impairments and yield reductions. Among them, low positive temperatures are of up most importance in tropical plant species, namely in Coffea spp. in which some acclimation ability has been reported. To further explain cold tolerance, the impacts on photosynthetic functioning and the expression of photosynthetic-related genes were analyzed. The experiments were carried out along a period of slow cold imposition (to allow acclimation), after chilling (4 °C) exposure and in the following rewarming period, using 1.5-year-old coffee seedlings of 5 genotypes with different cold sensitivity: Coffea canephora cv. Apoatã, Coffea arabica cv. Catuaí, Coffea dewevrei and 2 hybrids, Icatu (C. arabica × C. canephora) and Piatã (C. dewevrei × C. arabica). All genotypes suffered a significant leaf area loss only after chilling exposure, with Icatu showing the lowest impact, a first indication of a higher cold tolerance, contrasting with Apoatã and C. dewevrei. During cold exposure, net photosynthesis and Chl a fluorescence parameters were strongly affected in all genotypes, but stomatal limitations were not detected. However, the extent of mesophyll limitation, reflecting regulatory mechanisms and/or damage, was genotype dependent. Overnight retention of zeaxanthin was common to Coffea genotypes, but the accumulation of photoprotective pigments was highest in Icatu. That down-regulated photochemical events but efficiently protected the photosynthetic structures, as shown, e.g., by the lowest impacts on A_max and PSI activity and the strongest reinforcement of PSII activity, the latter possibly reflecting the presence of a photoprotective cycle around PSII in Icatu (and Catuaí). Concomitant to these protection mechanisms, Icatu was the sole genotype to present simultaneous upregulation of caCP22, caPI and caCytf, related to, respectively, PSII, PSI and to the complex Cytb₆/f, which could promote better repair ability, contributing to the maintenance of efficient thylakoid functioning. We conclude that Icatu showed the best acclimation ability among the studied genotypes, mostly due to a better upregulation of photoprotection and repair mechanisms. We confirmed the presence of important variability in Coffea spp. that could be exploited in breeding programs, which should be assisted by useful markers of cold tolerance, namely the upregulation of antioxidative molecules, the expression of selected genes and PSI sensitivity.