Chromoplast differentiation in bell pepper (Capsicum annuum) fruits.

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell pepper (Capsicum annuum) fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b6 f complex, the ATPase complex, and Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts' redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids, such as the oxidative pentose phosphate pathway (OPPP) and amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins decline but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation are consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper that lack a respiratory burst at the onset of fruit ripening.

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga.

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO2 ) fixation by toxic metals such as cadmium - classified as a priority pollutant - could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labelled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO2 fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (µXRF)/micro X-ray absorption near-edge structure (µXANES) at Cd LIII- edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the 13 C/12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.

MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain.

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

Synergistic Toxicity of Copper and Gold Compounds in Cupriavidus metallidurans.

The bacterium Cupriavidus metallidurans can reduce toxic gold(I/III) complexes and biomineralize them into metallic gold (Au) nanoparticles, thereby mediating the (trans)formation of Au nuggets. In Au-rich soils, most transition metals do not interfere with the resistance of this bacterium to toxic mobile Au complexes and can be removed from the cell by plasmid-encoded metal efflux systems. Copper is a noticeable exception: the presence of Au complexes and Cu ions results in synergistic toxicity, which is accompanied by an increased cytoplasmic Cu content and formation of Au nanoparticles in the periplasm. The periplasmic Cu-oxidase CopA was not essential for formation of the periplasmic Au nanoparticles. As shown with the purified and reconstituted Cu efflux system CupA, Au complexes block Cu-dependent release of phosphate from ATP by CupA, indicating inhibition of Cu transport. Moreover, Cu resistance of Au-inhibited cells was similar to that of mutants carrying deletions in the genes for the Cu-exporting PIB1-type ATPases. Consequently, Au complexes inhibit export of cytoplasmic Cu ions, leading to an increased cellular Cu content and decreased Cu and Au resistance. Uncovering the biochemical mechanisms of synergistic Au and Cu toxicity in C. metallidurans explains the issues this bacterium has to face in auriferous environments, where it is an important contributor to the environmental Au cycle.IMPORTANCEC. metallidurans lives in metal-rich environments, including auriferous soils that contain a mixture of toxic transition metal cations. We demonstrate here that copper ions and gold complexes exert synergistic toxicity because gold ions inhibit the copper-exporting P-type ATPase CupA, which is central to copper resistance in this bacterium. Such a situation should occur in soils overlying Au deposits, in which Cu/Au ratios usually are ≫1. Appreciating how C. metallidurans solves the problem of living in environments that contain both Au and Cu is a prerequisite to understand the molecular mechanisms underlying gold cycling in the environment, and the significance and opportunities of microbiota for specific targeting to Au in mineral exploration and ore processing.

A New Radio Frequency Plasma Oxygen Primary Ion Source on Nano Secondary Ion Mass Spectrometry for Improved Lateral Resolution and Detection of Electropositive Elements at Single Cell Level.

An important application field of secondary ion mass spectrometry at the nanometer scale (NanoSIMS) is the detection of chemical elements and, in particular, metals at the subcellular level in biological samples. The detection of many trace metals requires an oxygen primary ion source to allow the generation of positive secondary ions with high yield in the NanoSIMS. The duoplasmatron oxygen source is commonly used in this ion microprobe but cannot achieve the same quality of images as the cesium primary ion source used to produce negative secondary ions (C(-), CN(-), S(-), P(-)) due to a larger primary ion beam size. In this paper, a new type of an oxygen ion source using a rf plasma is fitted and characterized on a NanoSIMS50L. The performances of this primary ion source in terms of current density and achievable lateral resolution have been characterized and compared to the conventional duoplasmatron and cesium sources. The new rf plasma oxygen source offered a net improvement in terms of primary beam current density compared to the commonly used duoplasmatron source, which resulted in higher ultimate lateral resolutions down to 37 nm and which provided a 5-45 times higher apparent sensitivity for electropositive elements. Other advantages include a better long-term stability and reduced maintenance. This new rf plasma oxygen primary ion source has been applied to the localization of essential macroelements and trace metals at basal levels in two biological models, cells of Chlamydomonas reinhardtii and Arabidopsis thaliana.

High spatial resolution imaging of subcellular macro and trace element distribution during phagocytosis.

The bioavailability of trace elements in the course of evolution had an essential influence on the emergence of life itself. This is reflected in the co-evolution between eukaryotes and prokaryotes. In this study, the influence and cellular distribution of bioelements during phagocytosis at the host-pathogen interface were investigated using high-resolution nanoscale secondary ion mass spectrometry (NanoSIMS) and quantitative inductively coupled plasma mass spectrometry. In the eukaryotic murine macrophages (RAW 264.7 cell line), the cellular Fe/Zn ratio was found to be balanced, whereas the dominance of iron in the prokaryotic cells of the pathogen Salmonella enterica Serovar Enteritidis was ∼90% compared to zinc. This confirms the evolutionary increased zinc requirement of the eukaryotic animal cell. Using NanoSIMS, the Cs+ primary ion source allowed high spatial resolution mapping of cell morphology down to the subcellular level. At a comparable resolution, several low-abundant trace elements could be mapped during phagocytosis with a RF plasma O- primary ion source. An enrichment of copper and nickel could be detected in the prokaryotic cells. Surprisingly, an accumulation of cobalt in the area of the nuclear envelope was observed, indicating an interesting but still unknown distribution of this trace element in murine macrophages.

The pollen-specific class VIII-myosin ATM2 from Arabidopsis thaliana associates with the plasma membrane through a polybasic region binding anionic phospholipids.

Pollen tubes display polarized tip-growth and are a model to study the coordination of vesicular trafficking and cytoskeletal control. The molecular details of how dynamic actin filaments associate with the plasma membrane are currently unclear. In Arabidopsis thaliana, plasma membrane attachment of actin filaments may be mediated by four myosins representing the plant-specific myosin-subclass VIII, which localize to the plasma membrane and display only minor motor-activity. Here we explore the mode of membrane attachment of the pollen-expressed class VIII-myosins ATM2 and VIII-B through interaction with anionic membrane phospholipids. A fluorescent mCherry-ATM2-fusion decorated plasma membrane-peripheral actin filaments when expressed in tobacco pollen tubes, consistent with a role of class VIII-myosins at the membrane-cytoskeleton interface. As recombinant proteins, class VIII-myosins are prone to aggregation and to proteolysis, creating a challenge for their biochemical characterization. We describe a purification scheme for guanidinium chloride (GdmCl)-denatured recombinant proteins, followed by a renaturation protocol to obtain pure, soluble protein fragments of ATM2 and VIII-B. The fragments represent the C-terminal tail and coiled-coil-regions and lack the N-terminal actin-binding regions, IQ or motor domains. Based on lipid-overlays and liposome-sedimentation assays, the fragments of ATM2 and VIII-B bind anionic phospholipids. Small polybasic regions at the extreme C-termini were sufficient for lipid-binding of the respective protein fragments. When expressed in tobacco pollen tubes, a fluorescence-tagged variant of ATM2 lacking its lipid-binding region displayed substantially reduced plasma membrane association. The data indicate that class VIII-myosins may facilitate actin-plasma membrane attachment through interaction with anionic phospholipids, mediated by polybasic C-terminal lipid-binding domains.

Inhibitory effect of metals on animal and plant glutathione transferases.

Glutathione transferases (GSTs) represent a widespread enzyme superfamily in eukaryotes and prokaryotes catalyzing different reactions with endogenous and xenobiotic substrates such as organic pollutants. The latter are often found together with metal contamination in the environment. Besides performing of essential functions, GSTs protect cells by conjugation of glutathione with various reactive electrophiles. The interference of toxic metals with this functionality of GSTs may have unpredictable toxicological consequences for the organisms. In this review results from the recent literature are summarized and discussed describing the ability of metals to inhibit intracellular detoxification processes in animals and plants.

Mild proteasomal stress improves photosynthetic performance in Arabidopsis chloroplasts.

The proteasome is an essential protein-degradation machinery in eukaryotic cells that controls protein turnover and thereby the biogenesis and function of cell organelles. Chloroplasts import thousands of nuclear-encoded precursor proteins from the cytosol, suggesting that the bulk of plastid proteins is transiently exposed to the cytosolic proteasome complex. Therefore, there is a cytosolic equilibrium between chloroplast precursor protein import and proteasomal degradation. We show here that a shift in this equilibrium, induced by mild genetic proteasome impairment, results in elevated precursor protein abundance in the cytosol and significantly increased accumulation of functional photosynthetic complexes in protein import-deficient chloroplasts. Importantly, a proteasome lid mutant shows improved photosynthetic performance, even in the absence of an import defect, signifying that functional precursors are continuously degraded. Hence, turnover of plastid precursors in the cytosol represents a mechanism to constrain thylakoid membrane assembly and photosynthetic electron transport.

The rise and fall of major royal jelly proteins during a honeybee (Apis mellifera) workers' life.

The genome of the western honeybee (Apis mellifera) harbors nine transcribed major royal jelly protein genes (mrjp1-9) which originate from a single-copy precursor via gene duplication. The first MRJP was identified in royal jelly, a secretion of the bees' hypopharyngeal glands that is used by young worker bees, called nurses, to feed developing larvae. Thus, MRJPs are frequently assumed to mainly have functions for developing bee larvae and to be expressed in the food glands of nurse bees. In-depth knowledge on caste- and age-specific role and abundance of MRJPs is missing. We here show, using combined quantitative real-time PCR with quantitative mass spectrometry, that expression and protein amount of mrjp1-5 and mrjp7 show an age-dependent pattern in worker's hypopharyngeal glands as well as in brains, albeit lower relative abundance in brains than in glands. Expression increases after hatching until the nurse bee period and is followed by a decrease in older workers that forage for plant products. Mrjp6 expression deviates considerably from the expression profiles of the other mrjps, does not significantly vary in the brain, and shows its highest expression in the hypopharyngeal glands during the forager period. Furthermore, it is the only mrjp of which transcript abundance does not correlate with protein amount. Mrjp8 and mrjp9 show, compared to the other mrjps, a very low expression in both tissues. Albeit mrjp8 mRNA was detected via qPCR, the protein was not quantified in any of the tissues. Due to the occurrence of MRJP8 and MRJP9 in other body parts of the bees, for example, the venom gland, they might not have a hypopharyngeal gland- or brain-specific function but rather functions in other tissues. Thus, mrjp1-7 but not mrjp8 and mrjp9 might be involved in the regulation of phenotypic plasticity and age polyethism in worker honeybees.

The Effect of Diet on the Composition and Stability of Proteins Secreted by Honey Bees in Honey.

Honey proteins are essential bee nutrients and antimicrobials that protect honey from microbial spoilage. The majority of the honey proteome includes bee-secreted peptides and proteins, produced in specialised glands; however, bees need to forage actively for nitrogen sources and other basic elements of protein synthesis. Nectar and pollen of different origins can vary significantly in their nutritional composition and other compounds such as plant secondary metabolites. Worker bees producing and ripening honey from nectar might therefore need to adjust protein secretions depending on the quality and specific contents of the starting material. Here, we assessed the impact of different food sources (sugar solutions with different additives) on honey proteome composition and stability, using controlled cage experiments. Honey-like products generated from sugar solution with or without additional protein, or plant secondary metabolites, differed neither in protein quality nor in protein quantity among samples. Storage for 4 weeks prevented protein degradation in most cases, without differences between food sources. The honey-like product proteome included several major royal jelly proteins, alpha-glucosidase and glucose oxidase. As none of the feeding regimes resulted in different protein profiles, we can conclude that worker bees may secrete a constant amount of each bee-specific protein into honey to preserve this highly valuable hive product.

Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS.

Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1nm was used for imaging the ultrastructure of algae cells which have diameters of 5-10µm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1µm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast.

Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis.

Freshwater fungi which can survive under metal exposure receive increasing scientific attention. Enhanced synthesis of sulphide and glutathione but no phytochelatin synthesis in response to cadmium (up to 80 µM Cd(2+) in the medium) was measured in the aquatic hyphomycete Heliscus lugdunensis. Up to 25 µmol g(-1) dry mass the fungus formed sulphide in an exponentially Cd(2+)-concentration-dependent manner. Using light microscopy, precipitates were observed outside of the hyphae which could be determined as amorphous particles by X-ray diffraction (XRD). Energy dispersive X-ray spectroscopy (EDS) analysis indicated that these particles were mainly composed of Cd and S with an atomic ratio of 1:1, but some elements of the culture medium such as P and Cl were also present. Fungal cells exposed to Cd(2+) accumulated 12-28 µmol metal g(-1) dry mass over a period of 7-28 days. The results may indicate that sulphide could sequester excess Cd(2+) under oxygen deprived conditions and thereby reduce its toxicity via an additional avoidance mechanism of this fungus.

Identification of protein N-termini in Cyanophora paradoxa cyanelles: transit peptide composition and sequence determinants for precursor maturation.

Glaucophyta, rhodophyta, and chloroplastida represent the three main evolutionary lineages that diverged from a common ancestor after primary endosymbiosis. Comparative analyses between members of these three lineages are a rich source of information on ancestral plastid features. We analyzed the composition and the cleavage site of cyanelle transit peptides from the glaucophyte Cyanophora paradoxa by terminal amine labeling of substrates (TAILS), and compared their characteristics to those of representatives of the chloroplastida. Our data show that transit peptide architecture is similar between members of these two lineages. This entails a comparable modular structure, an overrepresentation of serine or alanine and similarities in the amino acid composition around the processing peptidase cleavage site. The most distinctive difference is the overrepresentation of phenylalanine in the N-terminal 1-10 amino acids of cyanelle transit peptides. A quantitative proteome analysis with periplasm-free cyanelles identified 42 out of 262 proteins without the N-terminal phenylalanine, suggesting that the requirement for phenylalanine in the N-terminal region is not absolute. Proteins in this set are on average of low abundance, suggesting that either alternative import pathways are operating specifically for low abundance proteins or that the gene model annotation is incorrect for proteins with fewer EST sequences. We discuss these two possibilities and provide examples for both interpretations.

Fungi from metal-polluted streams may have high ability to cope with the oxidative stress induced by copper oxide nanoparticles.

Increased commercialization of products based on metal oxide nanoparticles increases the likelihood that these nanoparticles will be released into aquatic environments, thus making relevant the assessment of their potential impacts on aquatic biota. Aquatic fungi are distributed worldwide and play a key role in organic matter turnover in freshwater ecosystems. The present study investigated the impacts of copper oxide spherical nanoparticles (CuO-NPs; <50 nm powder, 5 levels ≤200 mg/L) on cellular targets and antioxidant defenses in 5 fungal isolates collected from metal-polluted or nonpolluted streams. The CuO-NPs induced oxidative stress in aquatic fungi, as evidenced by intracellular accumulation of reactive oxygen species, and led to plasma membrane damage and DNA strand breaks in a concentration-dependent manner. Effects were more pronounced with a longer exposure time (3 d vs 10 d). Under CuO-NP exposure, mycelia of fungi collected from metal-polluted streams showed less oxidative stress and higher activities of superoxide dismutase and glutathione reductase compared with fungi from nonpolluted streams. The latter fungi responded to CuO-NPs with a stronger stimulation of glutathione peroxidase activity. These findings may indicate that fungi isolated from metal-polluted streams had a greater ability to maintain the pool of reduced glutathione than those from nonpolluted streams. Overall, results suggest that populations adapted to metals may develop mechanisms to cope with the oxidative stress induced by metal nanoparticles.

The zinc repository of Cupriavidus metallidurans.

Zinc is a central player in the metalloproteomes of prokaryotes and eukaryotes. We used a bottom-up quantitative proteomic approach to reveal the repository of the zinc pools in the proteobacterium Cupriavidus metallidurans. About 60% of the theoretical proteome of C. metallidurans was identified, quantified, and the defect in zinc allocation was compared between a ΔzupT mutant and its parent strain. In both strains, the number of zinc-binding proteins and their binding sites exceeded that of the zinc ions per cell, indicating that the totality of the zinc proteome provides empty binding sites for the incoming zinc ions. This zinc repository plays a central role in zinc homeostasis in C. metallidurans and probably also in other organisms.

Protein identification and quantification by data-independent acquisition and multi-parallel collision-induced dissociation mass spectrometry (MS(E)) in the chloroplast stroma proteome.

We report here a systematic evaluation of a multiplex mass spectrometry method coupled with ion mobility separation (HD-MS(E)) for the identification and quantification of proteins in the chloroplast stroma. We show that this method allows the robust quantification of reference proteins in mixtures, and it detects concentration differences with high sensitivity when three replicas are performed. Applied to the analysis of the chloroplast stroma proteome, HD-MS(E) identified and quantified many chloroplast proteins that were not previously identified in large-scale proteome analyses, suggesting HD-MS(E) as a suitable complementary tool for discovery proteomics. We find that HD-MS(E) tends to underestimate protein abundances at concentrations above 25fmol, which is likely due to ion transmission loss and detector saturation. This limitation can be circumvented by omitting the ion mobility separation step in the HD-MS(E) workflow. The robustness of protein quantification is influenced by the selection of peptides and their intensity distribution, therefore critical scrutiny of quantification results is required. Based on the HD-MS(E) quantification of chloroplast stroma proteins we performed a meta-analysis and compared published quantitative data with our results, using a parts per million normalization scheme. Important pathways in the chloroplast stroma show quantitative stability against different experimental conditions and quantification strategies. BIOLOGICAL SIGNIFICANCE: Our analysis establishes MS(E)-based Hi3 quantification as a tool for the absolute quantification of proteins in the chloroplast stroma. The meta-analysis performed with a parts per million normalization scheme shows that quantitative proteomics data acquired in different labs and with different quantification strategies yield comparable results for some metabolic pathways, while others show a higher variability. Our data therefore indicate that such meta-analyses allow distinguishing robust from fine-controlled metabolic pathways.

Physiological responses to nanoCuO in fungi from non-polluted and metal-polluted streams.

Nanocopper oxide (nanoCuO) is among the most widely used metal oxide nanoparticles which increases their chance of being released into freshwaters. Fungi are the major microbial decomposers of plant litter in streams. Fungal laccases are multicopper oxidase enzymes that are involved in the degradation of lignin and various xenobiotic compounds. We investigated the effects of nanoCuO (5 levels, ≤ 200 mg L(-1)) on four fungal isolates collected from metal-polluted and non-polluted streams by analyzing biomass production, changes in mycelial morphology, laccase activity, and quantifying copper adsorbed to mycelia, and ionic and nanoparticulate copper in the growth media. The exposure to nanoCuO decreased the biomass produced by all fungi in a concentration- and time-dependent manner. Inhibition of biomass production was stronger in fungi from non-polluted (EC50(10 days) ≤ 31 mg L(-1)) than from metal-polluted streams (EC50(10 days) ≥ 65.2 mg L(-1)). NanoCuO exposure led to cell shrinkage and mycelial degeneration, particularly in fungi collected from non-polluted streams. Adsorption of nanoCuO to fungal mycelia increased with the concentration of nanoCuO in the medium and was higher in fungi from non-polluted streams. Extracellular laccase activity was induced by nanoCuO in two fungal isolates in a concentration-dependent manner, and was highly correlated with adsorbed Cu and/or ionic Cu released by dissolution from nanoCuO. Putative laccase gene fragments were also detected in these fungi. Lack of substantial laccase activity in the other fungal isolates was corroborated by the absence of laccase-like gene fragments.

Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni.

Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

Identification of phytochelatins in the cadmium-stressed conjugating green alga Micrasterias denticulata.

Aquatic environments like peat bogs are affected by anthropogenic metal input into the environment. These ecosystems are inhabited by unicellular green algae of the class Zygnematophyceae. In this study the desmid Micrasterias denticulata was stressed with 600 nM Cd, 10 µM Cr and 300 nM Cu for 3 weeks. GSH levels were measured with HPLC and did not differ between the different treatments or the control. According to the metallo-thiolomics concept, mass spectrometry was used as a method for unambiguous thiol peptide identification. PC2, PC3 and PC4 were clearly identified in the Cd stressed sample with UPLC-MS by their MS spectrum and molecular masses. PC2 and PC3 were determined to be the main thiol compounds, while PC4 was only abundant in traces in Micrasterias. In addition, the identity of PC2 and PC3 was confirmed by MS/MS. No PCs were detected in the Cu stressed algae sample. However, in the Cr stressed sample traces of PC2 were indicated by a peak in UPLC-MS at the retention time of the PC2 standard, but the intensity was too low to acquire reliable MS and MS/MS spectra. In this study PCs have been detected for the first time in a green alga of the division Streptophyta, a close relative to higher plants.