Natural occurrence of fungal egg parasites of root-knot nematodes, Meloidogyne spp. in organic and integrated vegetable production systems in Spain

A survey was conducted to assess the biodiversity and frequency of infection of fungal egg parasites of Meloidogyne spp. and relate results to soil properties in organic and integrated vegetable production in Spain. Forty sites were sampled at the end of the cropping cycle, 30 under integrated and ten under organic production. Fungal egg parasites were isolated from all organically managed sites and from 73 % sites under integrated production. Species richness and Shannon–Wiener index did not differ between production systems but the percentage of fungal egg parasitism did, as well as soil properties. Percentages of egg parasitism higher than 40 % were found in five and three sites under organic and integrated production, respectively. In all these sites, Pochonia chlamydosporia was present alone or co-occurring with other fungal species. The relative frequency of P. chlamydosporia was positively related to the percentage of parasitism in both production systems.     

A quantitative analysis of the effect of nucleotides and the M domain on the association equilibrium of ClpB

ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.     

Evaluating microevolutionary models for the early settlement of the New World: the importance of recurrent gene flow with Asia

Different scenarios attempting to describe the initial phases of the human dispersal from Asia into the New World have been proposed during the last two decades. However, some aspects concerning the population affinities among early and modern Asians and Native Americans remain controversial. Specifically, contradictory views based mainly on partial evidence such as skull morphology or molecular genetics have led to hypotheses such as the "Two Waves/Components" and "Single Wave" or "Out of Beringia" model, respectively. Alternatively, an integrative scenario considering both morphological and molecular variation has been proposed and named as the "Recurrent Gene Flow" hypothesis. This scenario considers a single origin for all the Native Americans, and local, within-continent evolution plus the persistence of contact among Circum-Arctic groups. Here we analyze 2D geometric morphometric data to evaluate the associations between observed craniometric distance matrix and different geographic design matrices reflecting distinct scenarios for the peopling of the New World using basic and partial Mantel tests. Additionally, we calculated the rate of morphological differentiation between Early and Late American samples under the different settlement scenarios and compared our findings to the predicted morphological differentiation under neutral conditions. Also, we incorporated in our analyses some variants of the classical Single Wave and Two Waves models as well as the Recurrent Gene Flow model. Our results suggest a better explanatory performance of the Recurrent Gene Flow model, and provide additional insights concerning affinities among Asian and Native American Circum-Arctic groups.     

Draft genome sequence of the electricigen Acidiphilium sp. strain PM (DSM 24941)

Acidiphilium sp. strain PM (DSM 24941) was isolated from Rio Tinto's acidic, heavy metal-rich waters. Voltammetry experiments revealed that this strain is capable of electricity production even under aerobic conditions. Here we report the draft genome sequence of Acidiphilium sp. PM and a preliminary genome analysis that reveals a versatile respiratory metabolism.     

Ecological implications of unprecedented warm water anomalies on interannual prey preferences and foraging areas of Guadalupe fur seals

The Guadalupe fur seal (GFS) currently is recovering from near extinction and prey availability is an important factor. Scat and stable isotope analyses (SIA) were used to assess GFS foraging on Guadalupe Island during 2013–2016. This period was characterized by normal (2013) ocean temperatures followed by warm conditions (2014–2016). Scat samples (~40/year) were collected for prey identification. Additionally, lanugo samples from 1-month-old pups (50/year) were processed for SIA (δ¹³C/δ¹⁵N). Interannual prey preferences were identified (ANOSIM, p < .05), including significant differences between 2013 and the anomalous years. The most important prey species among these years was the jumbo squid; followed by the neon flying squid (2014–2016). The GFS diet was more specialized in 2013–2015 and more generalized in 2016. Prey from higher trophic levels (determined by scat analysis) were consumed during 2014–2016, but with significantly lower δ¹³C/δ¹⁵N values than in 2013 (ANOVA, p < .05 for both isotope ratios). This pattern may indicate more northerly or offshore foraging areas, with the presence of oceanic prey (neon flying squid). The widest isotopic niche was observed in 2015 (2.2‰²), reflecting a broader foraging area. Our findings are an important step toward better understanding the impacts of climate change on the recovery of GFS.     

Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g⁻¹ protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0–55 000 units g⁻¹ protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.Microorganisms play a crucial role in soil genesis by facilitating mineralization not only for soil organic matter (Leigh Mascarelli, 2009) but also for prevalent and persistent pollutants, such as polyaromatic hydrocarbons (PAH) and heterocyclic aromatic compounds (Lu et al., 2011). Such compounds are common additives in crude oil and industrial chemical products, such as dyes, flavouring compounds, plasticizers, perfumes, pesticides and insect repellent as well as, more recently, microelectronics, printed circuit boards, silk screen printing devices, optical disks and black colour tube matrices (Kästner, 2000; Chae et al., 2002). Multiple microorganisms can obtain energy from such hydrophobic aromatics (Lu et al., 2011), which are primarily released into the environment through anthropogenic activities. When bacteria are confronted with aromatic compounds, the cells encounter an interesting contradiction. On the one hand, such chemical species can be mineralized to yield carbon and energy for growth, which allows microorganisms to colonize niches that are refractory to other microbes (Dominguez-Cuevas et al., 2006; Lu et al., 2011). On the other hand, aromatic compounds over a certain threshold are toxic for bacteria because they partition and disorganize the cell membrane by removing lipids and proteins, which leads to cell death (Sikkema et al., 1995; von Wallbrunn et al., 2003). To cope with such activities, bacteria have developed multiple chemical tolerance mechanisms and an extensive enzyme arsenal (e.g. oxygenases, O-demetylases, CoA synthases and ligases, aldolases, alcohol dehydrogenases and α/β hydrolases) for mineralizing aromatic compounds by submitting them to the central metabolism (Pérez-Pantoja et al., 2008; 2009; Vilchez-Vargas et al., 2013).Recent studies suggest that multifunctional esterase/lipase-like proteins from the α/β hydrolase family that can hydrolyse both C-C and C-O bonds may exist in nature at much higher levels than previously thought (Alcaide et al., 2013). From an ecological perspective, such proteins may contribute to global carbon cycling processes for complex substrates, including recalcitrant organic pollutants. From a biotechnological perspective, such proteins may open unexpected research avenues for biotechnology applications. Esterases/lipases from the α/β hydrolase family have demonstrated such activities for catechol and biphenyl derivatives (Alcaide et al., 2013). However, the potential implication for the degradation of and biotechnology of complex organic molecules with two or more aromatic rings, specifically benzene rings (polyaromatic hydrocarbons – PAH), has not been described, even though the PAH degradation phenomenon is relatively well known (Pérez-Pantoja et al., 2008; 2009; Seo et al., 2009). Such activities are especially significant because the chemistry for PAH esters has been a subject of considerable interest due to their chemical and physical properties (e.g. polymers produced with versatile photo-reactivities) (Noh et al., 2001; Chae et al., 2002; Zhu et al., 2011). Heterogeneous catalysts have successfully been used to produce PAH-like (naphthalene, anthracene, phenanthrene, benzyl and phthalate) derivatives (Noh et al., 2001; Zhu et al., 2011; Maruyama et al., 2012). However, to the best of our knowledge, no study in the specialized literature has involved identification of potential applications for esterase/lipase-like proteins as catalysts for modifying PAHs.Because contaminated environments and microbial communities derived therefrom are an excellent source for enzymes that act on aromatics (Pérez-Pantoja et al., 2008; 2009; Guazzaroni et al., 2013), we now ask whether such α/β hydrolases can be identified therein. We generated a subset of 5500 clones (in Escherichia coli EPI300-T1^R; Epicentre Biotechnologies; Madison, WI, USA; Alcaide et al., 2013) from a naphthalene-enriched community (CN1) derived from PAH-contaminated soil (Guazzaroni et al., 2013), which included nearly 0.17 Gbp of community genomes, that were evaluated for their ability to hydrolyse α-naphthyl acetate (Reyes-Duarte et al., 2012). Four unique positive clones (hit rate 1:1375) were identified as active, and one (herein, CN1E1) was selected for in-depth analysis due to its high activity (halo/colour formation) and capacity for hydrolysis in agar-based assays using the model PAH ester naphthalene carboxylic acid methyl ester (Fig. 1). The insert [30 164 bp; nucleotide sequence available at the National Center for Biotechnology Information (NCBI) under accession number SRP030024] was sequenced [Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT, USA) at LifeSequencing S.L., Valencia, Spain], analysed and compared with the sequences in the NCBI non-redundant public database (Altschul et al., 1997). Thirty-one predicted open reading frames were identified (Lukashin and Borodovsky, 1998); one encoded a putative esterase (CN1E1) with the α/β hydrolase fold. The protein [311 amino acids (AA); molecular weight (MW) 33 146 Da; isoelectric point (pI) 6.34] was produced in a soluble form (Fig. 2) upon expression in the pET-46 Ek/LIC vector (using the forward and reverse primers 5′-GACGACGACAAGATGGCGGTAGATCCG-3′ and 5′-GAGGAGAAGCCCGGTTATCTCGGTCCGGC-3′ respectively) and E. coli BL21 (DE3) (conditions described in Alcaide et al., 2013). The sequence was analysed, which indicated that it belonged to the α/β hydrolase superfamily; the esterase/lipase-like protein from Paenibacillus mucilaginosus 3016 ({"type":"entrez-protein","attrs":{"text":"YP_005313749.1","term_id":"379721618","term_text":"YP_005313749.1"}}YP_005313749.1) is the most similar enzyme (AA sequence identity: 54%; similarity: 68%). It was also homologous (≤ 56% identity) to predicted esterases/lipases from uncultured microorganisms derived from soil and marine sediment samples (Fig. 3). This enzyme can be categorized in the microbial family IV described by Arpigny and Jaeger (1999); it includes a typical -GlyxSerxGly-motif and presumptive Ser-Asp-His catalytic triad (Ser155, Asp250 and His280). CN1E1 was structurally most similar to a lipase/esterase-like protein from Alicyclobacillus acidocaldarius [identity: 45%; Protein Data Bank (PDB) code 2HM7_A].Open in a separate windowFigure 1Hydrolytic phenotype for E. coli BL21 (DE3) that either expressed or did not express CN1E1 using the pET-46 Ek/LIC vector. The cells were plated on fresh Luria Bertani (LB) plates with ampicillin (50 μg ml⁻¹). The plates [with 1.0 mM isopropyl-β-D-galactopyranoside (IPTG)] were incubated for 12 h at 37°C and then covered with a second layer that included the substrate [20 ml 5 mM N-(2-hydroxyethyl) piperazine-N′-(3-propanesulfonic acid) (EPPS) buffer, pH 8.0, 0.4% agarose and 320 μl of a naphthalene carboxylic acid methyl ester solution in acetonitrile (100 mg ml⁻¹)]. The active phenotype was apparent due to the colour change resulting from substrate hydrolysis and acetic acid formation, which changes the pH indicator from red to yellow.Open in a separate windowFigure 2A Coomassie-stained SDS-PAGE gel showed that the active form of the protein CN1E1 was over-expressed in E. coli at 16°C. The expression and purification conditions were reported in Martínez-Martínez and colleagues (2013). As shown, a high percentage of the protein was produced in its soluble form, which yielded a purity greater than 98% after a single His₆-tag purification step. Abbreviation: MW, molecular weight marker. The lanes include the following: lane 1, soluble cell fraction after induction with 1.0 mM IPTG; lane 2, soluble cell fraction without IPTG; lane 3, molecular weight marker (from top to bottom: 225, 150, 100, 75, 50, 35, 25 and 15 kDa); and lane 4, pure protein after His₆-tag purification.Open in a separate windowFigure 3The unrooted circular neighbour-joining tree indicates the polypeptide sequence phylogenetic positions for the CN1E1 enzyme (in boldface) and reference hydrolases. The tree was constructed using an aligned 297 AA-long sequence. The GenBank and PDB accession numbers are indicated in brackets. For the dendrogram construction details, see Martínez-Martínez and colleagues (2013) and Tamura and colleagues (2007). The Family V cluster includes the sequences with the following accession numbers: YP960710.1, {"type":"entrez-protein","attrs":{"text":"ADP98993.1","term_id":"311696120","term_text":"ADP98993.1"}}ADP98993.1, ZP01735705.1, ZP01735705.1, {"type":"entrez-protein","attrs":{"text":"CAE54381.1","term_id":"50871368","term_text":"CAE54381.1"}}CAE54381.1, ZP01307774.1, {"type":"entrez-nucleotide","attrs":{"text":"X53869","term_id":"44522","term_text":"X53869"}}X53869, {"type":"entrez-protein","attrs":{"text":"AEO74498.1","term_id":"347304384","term_text":"AEO74498.1"}}AEO74498.1, YP001347584.1, NP251639.1, YP790224.1, YP0029804424.1, YP002909304.2, YP001810250.1, ZP03570306.1, YP299126.1, YP583166.1, YP002005156.1, 725653.1 and YP006884923.1. The Family VI cluster includes YP007541785, WP007625024, WP008294645, S78600 and PDB 3CN7. The scale bar represents 0.2 substitutions per position. The lipase/esterase families are depicted based on the Arpigny and Jaeger (1999) classification system. The abbreviations are as follows: EST, esterase; LIP, lipase; HYP, hypothetical protein; ACTIN, Actinobacteria; FIR, Firmicutes; PLANCT, Planctomycetes; PROT, Proteobacteria; UNCB, uncultured bacterium.The pure enzyme was most active at 25–30°C and pH 8.5 (Fig. 4); it retained approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. The substrates used herein included 12 model esters [6 p-nitrophenol (pNP) and 6 triacylglycerol] and 86 structurally different esters (Martínez-Martínez et al., 2013). Based on the specific activities determined (units g⁻¹; one unit is the amount of enzyme that hydrolyses 1 μmol of substrate per min under the assay conditions, as reported by Martínez-Martínez et al., 2013), CN1E1 showed a capacity for accepting 61 esters with different alcohol and acid moieties (Fig. 5). Considering the acyl chain length and p-nitrophenyl ester hydrolysis, CN1E1 was most active with pNP-acetate (∼ 55 000 units g⁻¹ at 40°C and pH 8.0); it also hydrolysed pNP-dodecanoate, albeit at a rate of four orders of magnitude lower. As shown in Fig. 5, pNP esters were the preferred substrates among the examined esters. This enzyme hydrolysed short-chain triacylglycerols that ranged from triacetin (which was preferred; ∼ 23 000 units g⁻¹ at 40°C and pH 8.0) to tricaproin (∼ 21 units g⁻¹) and short-chain halogenated and non-halogenated alkyl and aryl esters; methyl bromoacetate (∼ 31 000 units g⁻¹) and butyl acetate (∼ 11 700 units g⁻¹) were preferred respectively (Fig. 5). Because the enzyme preferred short-chain triacylglycerols and short-to-medium size alkyl and aryl esters, the α/β-hydrolase is likely an esterase. It also showed a capacity for accepting tri-O-acetyl-glucal (∼ 12 500 units g⁻¹), the carbohydrate ester α-D-glucose pentaacetate (∼ 373 units g⁻¹) and hydroxycinnamic acid-like esters, such as 2,5-dihydroxycinnamic acid methyl ester (∼ 10 700 units g⁻¹), methyl cinnamate (∼ 28 units g⁻¹) and caffeic acid phenethyl ester (∼ 19 units g⁻¹) (Fig. 5). Such characteristics suggest that the enzyme can support polysaccharide degradation. Amino acid esters, such as L-proline and L-serine esters, were also hydrolysed (∼ 24–1.2 units g⁻¹); however, they were among the non-preferred substrates (Fig. 5). Under our assay conditions, the esterase was also active (from ∼ 609 to 5.2 units g⁻¹) and enantio-selective for (±)-menthyl-acetate, (±)-methyl lactate, (±)-neomenthyl-acetate, (±)-methyl-mandelate and (±)-glycidyl 4-nitrobenzoate with an (S)-preference (Fig. 5). γ-Butyrolactone (∼ 141 units g⁻¹), γ-valerolactone (∼ 81 units g⁻¹) and (±)-pantolactone [the (R) enantiomer was preferred; ∼ 606 units g⁻¹] were also accepted as substrates (Fig. 5).Open in a separate windowFigure 4The wild-type CN1E1 α/β hydrolase pH (A) and temperature (B) profiles. Esterase activity using pNP-propionate (at 410 nm) was determined as described previously (Martínez-Martínez et al., 2013). The standard deviation (SD) for the triplicate assays is shown. We examined pH values between 4.0 and 9.5 and temperatures between 4 and 80°C to determine the optimal parameters. The following buffers were examined at 40 mM: sodium citrate (pH 4.0–4.5), sodium acetate (pH 5.0–6.0), 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5–6.0), piperazine-N,N′-bis(ethanesulfonic acid) (PIPES) (pH 6.0–7.0), 4-(2-hydroxyethylpiperazine-1-ethanesulfonic acid (HEPES) (pH 7.0–8.0), K/Na-phosphate (pH 7.5), Tris-HCl (pH 8.5) and glycine (pH 9.0–9.5). The pH was adjusted at 25°C. The pH and temperature profiles were collected at 40°C (panel A) and pH 7.0 (using 40 mM HEPES; panel B) respectively. In panels (A) and (B), 100% of the activity refers to 27.08 ± 1.80 and 21.17 ± 1.41 units mg⁻¹ respectively.Open in a separate windowFigure 5Substrate profile for the wild-type CN1E1 α/β hydrolase using a set of structurally diverse esters. The specific activities were calculated in triplicate as described by Martínez-Martínez and colleagues (2013) at 40°C in 20 mM HEPES buffer pH 7.0 (for pNP esters) or 5 mM EPPS buffer pH 8.0 (for the remaining esters). The standard deviation (SD) for the triplicate assays is shown. The chemicals used for the enzymatic experiments were the purest grade available and were purchased from Fluka-Aldrich-Sigma Chemical Co. (St. Louis, MO, USA). The hydroxycinnamic-like esters were supplied by Apin Chemicals (Oxon, UK), the methyl phenanthrene-3-carboxylate and methyl phenanthrene-9-carboxylate were supplied by Wuhan Farthest Chemical (Mainland, China), and the anthracene-9-carboxylic acid methyl ester and anthracene-3-carboxylic acid methyl ester were obtained from Alfa Aesar (Karlsruhe, Germany). Insets (A) to (C) in the figure represent a zoom for substrates hydrolysed at low rates.Because the active clone with CN1E1 actively hydrolysed naphthalene carboxylic acid methyl ester on an agar plate (Fig. 1), we further tested and quantified hydrolysis for the pure enzyme using this and other model PAH esters; the pH-indicator protocol (at 40°C and pH 8.0) used to examine hydrolysis for the above esters was also used for this experiment (Martínez-Martínez et al., 2013). CN1E1 showed broad reactivity towards PAH-like esters, including phthalate alkyl and aryl (benzene) esters (from ∼ 21 000 to 6100 units g⁻¹), alkyl esters of phenanthrene (from ∼ 20 500 to 7600 units g⁻¹), anthracene (from ∼ 18 400 to 11 000 units g⁻¹), naphthalene (from ∼ 10 000 to 7800 units g⁻¹) and protocatechuate (∼ 8800 units g⁻¹) and benzoate esters with substituent phenyl/benzene rings (from ∼ 7600 to 7200 units g⁻¹). The majority of such compounds were among the best substrates for CN1E1 (Fig. 5); this enzyme showed regio-selectivity for phenanthrene-3-carboxylate over methyl phenanthrene-9-carboxylate (2.7-fold) and, to a lesser extent, for anthracene-2-carboxylate over anthracene-9-carboxylate (1.7-fold). Although previous reports have shown that other esterases can degrade phthalate esters (Maruyama et al., 2005; Saito et al., 2010; Wu et al., 2013), to the best of our knowledge, the specialized literature has not reported an esterase with the capacity to degrade PAH esters with multiple aromatic (e.g. benzene) rings.The half-saturation (Michaelis) coefficient (K_m), catalytic rate constant (k_cat) and catalytic efficiency (k_cat/K_m) values were determined for the 14 best and structurally distinct substrates (2013) at pH 8.0 and 40°C (except for pNP-acetate were pH 7.0 was used to ensure substrate stability). The substrates analysed include pNP-acetate (the model p-nitrophenyl ester), triacetin (the model triacylglycerol), methyl bromoacetate (the model halogenated ester), tri-O-acetyl-glucal (the model polysaccharide-like ester), butyl acetate (the model alkyl ester), phenyl acetate (the model aryl ester), 2,5-dihydroxycinnamic acid methyl ester (the model hydroxycinnamic-like ester) and seven chemically distinct model PAH esters. As shown in 2013)

Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from the Wosera region of Papua New Guinea

The merozoite surface protein 2 (MSP2) is a leading asexual-stage malaria vaccine candidate that has already proven to have an effect in phase I/IIb vaccine trials, where it was tested in combination with other antigens. Alleles of msp2 fall within two major allelic families, 3D7 and FC27. We analyzed the msp2 genotype in 306 asymptomatic and 63 symptomatic infections from the Wosera region of Papua New Guinea. The multiplicity of infection and the distribution of msp2 alleles was similar to that found in previous studies in the region, but there was no association found between FC27-type or 3D7-type forms of MSP2 and clinical malaria.     

Reactive oxygen species as transducers of sphinganine-mediated cell death pathway

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.^1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.^2,3 This defense strategy has been known as the hypersensitive response (HR).^4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).^1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.⁶Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.^1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.² Although ROS have been identified downstream of LCBs in the route towards PCD,¹ the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.⁷Similar to what is observed in pathogen defense,³ increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.^1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.^1,2,9–11An early production of ROS has been linked to an increase of LCBs. For example, an H₂O₂ burst is found in tobacco cells after 2–20 min of sphinganine supplementation,¹² and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H₂O₂ were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.¹ However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,^13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,¹⁵ or to intracellular ROS that diffuse outwards.^16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references ²⁸ and ²⁹. DPI was used at 50 µM. (B) H₂O₂ formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H₂O₂ according to Thordal-Christensen et al.³⁰It has been suggested that the H₂O₂ burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.¹² It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.⁹ In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.¹⁵ This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.² All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H₂O₂ formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H₂O₂ with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis¹⁸ and the mutant has a FB1-resistant phenotype.² These results indicate that mutations in the LCB1¹ and LCB2a² genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H₂O₂. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.³ In this condition, we clearly detected H₂O₂ formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.² Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H₂O₂ formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.²Open in a separate windowFigure 2Conditions of LCBs elevation produce H₂O₂ formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H₂O₂ formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 10⁸ UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H₂O₂ formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl₂ and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.² Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H₂O₂ produce alterations in mitochondrial morphology.^20–22 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.^22–24It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.^25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.²⁶ At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD²⁷ as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.     

Emergent macrophytes act selectively on ammonia-oxidizing bacteria and archaea

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were quantified in the sediments and roots of dominant macrophytes in eight neutral to alkaline coastal wetlands. The AOA dominated in most samples, but the bacterial-to-archaeal amoA gene ratios increased with increasing ammonium levels and pH in the sediments. For all plant species, the ratios increased on the root surface relative to the adjacent bulk sediment. This suggests that root surfaces in these environments provide conditions favoring enrichment of AOB.