The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Volatilome and proteome responses to Colletotrichum lindemuthianum infection in a moderately resistant and a susceptible bean genotype.

We analyzed the changes in the volatilome, proteome, stomatal conductance, salicylic and jasmonic acid contents of a susceptible and a moderately resistant genotype of common bean, Phaseoulus vulgaris L., challenged with Colletotrichum lindemuthianum, the causal agent of fungal anthracnose. Our results indicate differences at both proteome and volatilome levels between the two genotypes, before and after the infection, and different defense strategies. The moderately resistant genotype hindered pathogen infection, invasion, and replication mainly by maintaining epidermal and cell wall structure. The susceptible genotype was not able to limit the early stages of pathogen infection. Rather, stomatal conductance increased in the infected susceptible genotype, and enhanced synthesis of Green Leaf Volatiles and salicylic acid was observed, together with a strong hypersensitive response. Proteomic investigation provided a general framework for physiological changes, whereas observed variations in the volatilome suggested that volatile organic compounds may principally represent stress markers rather than defensive compounds per se.

Quantitative phosphoproteomics reveals novel roles of cAMP in plants.

3',5'-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3',5'-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular "sponge" that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.

A multifaceted approach to reveal the very-fine root's response of Fagus sylvatica seedlings to different drought intensities.

How temperate trees respond to drier summers strongly depends on the drought susceptibility and the starch reserve of the very-fine roots (<0.5 mm in diameter). We performed morphological, physiological, chemical, and proteomic analyses on very-fine roots of Fagus sylvatica seedlings grown under moderate- and severe drought conditions. Moreover, to reveal the role of the starch reserves, a girdling approach was adopted to interrupt the flux of photosynthates toward the downstream sinks. Results show a seasonal sigmoidal growth pattern without evident mortality under moderate drought. After the severe-drought period, intact plants showed lower starch concentration and higher growth than those subjected to moderate drought, highlighting that very-fine roots rely on their starch reserves to resume growth. This behavior caused them to die with the onset of autumn, which was not observed under moderate drought. These findings indicated that extreme dry soil conditions are needed for significant root death in beech seedlings and that mortality mechanisms are defined within individual compartments. The girdling treatment showed that the physiological responses of very-fine roots to severe drought stress are critically related to the altered load or the reduced transport velocity of the phloem and that the changes in starch allocation critically alter the distribution of biomass. Proteomic evidence revealed that the phloem flux-dependent response was characterized by the decrease of carbon enzymes and the establishment of mechanisms to avoid the reduction of the osmotic potential. The response independent from the aboveground mainly involved the alteration of primary metabolic processes and cell wall-related enzymes.

Proteomic characterization of Shiitake (Lentinula edodes) post-harvest fruit bodies grown on hardwood logs and isolation of an antibacterial serine protease inhibitor.

Lentinula edodes (Shiitake) is one of the most heavily cultivated mushrooms in the world with proven antioxidant and antibacterial properties, among others. Evidence indicates that the choice of mushroom cultivation technique strongly influences the production of bioactive compounds, but to date the nature of many of these compounds has not been fully established. This work focuses on the proteomic characterization of L. edodes to highlight the main active processes two days after harvest and elucidates the proteins involved in the known antioxidant and antibacterial proprieties of Shiitake fruit bodies cultivated on oak logs. A label-free approach allowed us to identify a total of 2702 proteins which were mainly involved in carbohydrate and protein metabolism, cell growth and replication, indicating that several developmental processes remain active in fruit bodies post-harvest. Proteins with antioxidant activities were identified, indicating the contribution of proteins to the antioxidant properties of L. edodes extracts. Antibacterial assays also reveal the activity of a serine protease inhibitor that strongly accumulates in the post-harvest fruit body grown on oak logs. Overall, this study contributes to the understanding of the impact of the log cultivation method on the production of Shiitake mushrooms richest in high-value bioactive compounds.

Proteomics as a tool to decipher plant responses in arbuscular mycorrhizal interactions: a meta-analysis.

The beneficial symbiosis between plants and arbuscular mycorrhizal (AM) fungi leads to a deep reprogramming of plant metabolism, involving the regulation of several molecular mechanisms, many of which are poorly characterized. In this regard, proteomics is a powerful tool to explore changes related to plant-microbe interactions. This study provides a comprehensive proteomic meta-analysis conducted on AM-modulated proteins at local (roots) and systemic (shoots/leaves) level. The analysis was implemented by an in-depth study of root membrane-associated proteins and by a comparison with a transcriptome meta-analysis. A total of 4262 differentially abundant proteins were retrieved and, to identify the most relevant AM-regulated processes, a range of bioinformatic studies were conducted, including functional enrichment and protein-protein interaction network analysis. In addition to several protein transporters which are present in higher amounts in AM plants, and which are expected due to the well-known enhancement of AM-induced mineral uptake, our analysis revealed some novel traits. We detected a massive systemic reprogramming of translation with a central role played by the ribosomal translational apparatus. On one hand, these new protein-synthesis efforts well support the root cellular re-organization required by the fungal penetration, and on the other they have a systemic impact on primary metabolism.

Isoprene Emission Influences the Proteomic Profile of Arabidopsis Plants under Well-Watered and Drought-Stress Conditions.

Isoprene is a small lipophilic molecule synthesized in plastids and abundantly released into the atmosphere. Isoprene-emitting plants are better protected against abiotic stresses, but the mechanism of action of isoprene is still under debate. In this study, we compared the physiological responses and proteomic profiles of Arabidopsis which express the isoprene synthase (ISPS) gene and emit isoprene with those of non-emitting plants under both drought-stress (DS) and well-watered (WW) conditions. We aimed to investigate whether isoprene-emitting plants displayed a different proteomic profile that is consistent with the metabolic changes already reported. Only ISPS DS plants were able to maintain the same photosynthesis and fresh weight of WW plants. LC-MS/MS-based proteomic analysis revealed changes in protein abundance that were dependent on the capacity for emitting isoprene in addition to those caused by the DS. The majority of the proteins changed in response to the interaction between DS and isoprene emission. These include proteins that are associated with the activation of secondary metabolisms leading to ABA, trehalose, and proline accumulations. Overall, our proteomic data suggest that isoprene exerts its protective mechanism at different levels: under drought stress, isoprene affects the abundance of chloroplast proteins, confirming a strong direct or indirect antioxidant action and also modulates signaling and hormone pathways, especially those controlling ABA synthesis. Unexpectedly, isoprene also alters membrane trafficking.

Proteomic analysis reveals how pairing of a Mycorrhizal fungus with plant growth-promoting bacteria modulates growth and defense in wheat.

Plants rely on their microbiota for improving the nutritional status and environmental stress tolerance. Previous studies mainly focused on bipartite interactions (a plant challenged by a single microbe), while plant responses to multiple microbes have received limited attention. Here, we investigated local and systemic changes induced in wheat by two plant growth-promoting bacteria (PGPB), Azospirillum brasilense and Paraburkholderia graminis, either alone or together with an arbuscular mycorrhizal fungus (AMF). We conducted phenotypic, proteomic, and biochemical analyses to investigate bipartite (wheat-PGPB) and tripartite (wheat-PGPB-AMF) interactions, also upon a leaf pathogen infection. Results revealed that only AMF and A. brasilense promoted plant growth by activating photosynthesis and N assimilation which led to increased glucose and amino acid content. The bioprotective effect of the PGPB-AMF interactions on infected wheat plants depended on the PGPB-AMF combinations, which caused specific phenotypic and proteomic responses (elicitation of defense related proteins, immune response and jasmonic acid biosynthesis). In the whole, wheat responses strongly depended on the inoculum composition (single vs. multiple microbes) and the investigated organs (roots vs. leaf). Our findings showed that AMF is the best-performing microbe, suggesting its presence as the crucial one for synthetic microbial community development.

Label-Free Proteomic Approach to Study the Non-lethal Effects of Silver Nanoparticles on a Gut Bacterium.

Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs) have become one of the most commonly utilized because of their antimicrobial properties. Despite their common use, the effects of sublethal concentrations of AgNPs, especially on gut biofilms, have been poorly investigated. To address this issue, we investigated in vitro the proteomic response of a monospecies Escherichia coli gut biofilm to chronic and acute exposures in sublethal concentrations of AgNPs. We used a new gel- and label-free proteomic approach based on shotgun nanoflow liquid chromatography-tandem mass spectrometry. This approach allows a quantification of the whole proteome at a dynamic range that is higher than the traditional proteomic investigation. To assess all different possible exposure scenarios, we compared the biofilm proteome of four treatments: (i) untreated cells for the control treatment, (ii) cells treated with 1 µg/ml AgNPs for 24 h for the acute treatment, (iii) cells grown with 1 µg/ml AgNPs for 96 h for the chronic treatment, and (iv) cells grown in the presence of 1 µg/ml AgNPs for 72 h and then further treated for 24 h with 10 µg/ml AgNPs for the chronic + acute treatment. Among the 1,917 proteins identified, 212 were significantly differentially expressed proteins. Several pathways were altered including biofilm formation, bacterial adhesion, stress response to reactive oxygen species, and glucose utilization.

Proteasome-mediated remodeling of the proteome and phosphoproteome during kiwifruit pollen germination.

Proteasome activity is essential for pollen tube emergence and growth; nevertheless, little is known about proteasome function at the molecular level. The objective of this study was to identify molecular targets and pathways which are directly/indirectly controlled by the proteasome during pollen germination. To this aim, changes in the proteome and phosphoproteome of Actinidia pollen, germinated in the presence of the proteasome inhibitor MG132, were investigated. Phosphoproteins were enriched by metal oxide/hydroxide affinity chromatography and phosphopeptides were further isolated using titanium ion (Ti4+) functional magnetic microparticles prior to liquid chromatography-tandem mass spectrometry analysis. Our results show that proteasome inhibition affects the phosphoproteome more profoundly than the proteome. Accordingly, the steady-state abundance of some kinases and phosphatases was changed and/or their phosphorylation status altered. Notably, affected proteins are involved in processes that are fundamental to pollen germination such as cytoskeletal organization, vesicular transport, cell wall synthesis and remodeling, protein synthesis, folding and degradation as well as energetic metabolism. Our data provide a molecular framework for the structural alterations observed when the proteasome is inhibited, contribute to the understanding of how proteasome activity regulates pollen germination, show the cross-talk between phosphorylation and proteasomal degradation and are a resource for further functional analyses. SIGNIFICANCE: Pollen germination and tube growth are fundamental to successful fertilization in seed plants. These events are based on dramatic remodeling and the dismantling of existing programs, which are replaced by new ones. Degradation plays a prominent role in reshaping the protein repertoire, also cross talking with the bulk of post-translational modifications. At present, phosphorylation is the only modification studied in germinating pollen on a large scale. The proteasome has been universally recognized as one of the most important sites for protein degradation and its function has been shown to be essential for pollen tube emergence and elongation. Upon proteasome inhibition structural alterations and dysregulation of pivotal processes governing pollen germination have been described; however, a mechanistic framework for the proteasome function at the molecular level is still lacking. In this investigation we provide the very first view of the global impact of the proteasome in remodeling the proteome and phosphoproteome during germination and tube growth. Our results show how proteasome inhibition alters the levels, and profoundly affects the phosphorylation status of many proteins involved, controlling energetic and synthetic pathways and signaling cascades.

Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat.

Besides improved mineral nutrition, plants colonised by arbuscular mycorrhizal (AM) fungi often display increased biomass and higher tolerance to biotic and abiotic stresses. Notwithstanding the global importance of wheat as an agricultural crop, its response to AM symbiosis has been poorly investigated. We focused on the role of an AM fungus on mineral nutrition of wheat, and on its potential protective effect against Xanthomonas translucens. To address these issues, phenotypical, molecular and metabolomic approaches were combined. Morphological observations highlighted that AM wheat plants displayed an increased biomass and grain yield, as well as a reduction in lesion area following pathogen infection. To elucidate the molecular mechanisms underlying the mycorrhizal phenotype, we investigated changes of transcripts and proteins in roots and leaves during the double (wheat-AM fungus) and tripartite (wheat-AM fungus-pathogen) interaction. Transcriptomic and proteomic profiling identified the main pathways involved in enhancing plant biomass, mineral nutrition and in promoting the bio-protective effect against the leaf pathogen. Mineral and amino acid contents in roots, leaves and seeds, and protein oxidation profiles in leaves, supported the omics data, providing new insight into the mechanisms exerted by AM symbiosis to confer stronger productivity and enhanced resistance to X. translucens in wheat.

Effect of Inulin on Proteome Changes Induced by Pathogenic Lipopolysaccharide in Human Colon.

In the present study, the protective role of inulin against lipopolysaccharide (LPS)-induced oxidative stress was evaluated on human colonic mucosa using a proteomic approach. Human colonic mucosa and submucosa were sealed between two chambers, with the luminal side facing upwards and overlaid with Krebs (control), LPS or LPS+ inulin IQ solution. The solutions on the submucosal side (undernatants) were collected following 30 min of mucosal exposure. iTRAQ based analysis was used to analyze the total soluble proteomes from human colonic mucosa and submucosa treated with different undernatants. Human colonic muscle strips were exposed to the undernatants to evaluate the response to acetylcholine. Inulin exposure was able to counteract, in human colonic mucosa, the LPS-dependent alteration of some proteins involved in the intestinal contraction (myosin light chain kinase (MLCK), myosin regulatory subunit (MYL)), to reduce the up-regulation of two proteins involved in the radical-mediated oxidative stress (the DNA-apurinic or apyrimidinic site) lyase) APEX1 and the T-complex protein 1 subunit eta (CCT7) and to entail a higher level of some detoxification enzymes (the metallothionein-2 MT2A, the glutathione-S-transferase K GSTk, and two UDP- glucuronosyltransferases UGT2B4, UGT2B17). Inulin exposure was also able to prevent the LPS-dependent intestinal muscle strips contraction impairment and the mucosa glutathione level alterations. Exposure of colonic mucosa to inulin seems to prevent LPS-induced alteration in expression of some key proteins, which promote intestinal motility and inflammation, reducing the radical-mediated oxidative stress.

An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts.

Arbuscular mycorrhizal fungi (AMF) are obligate plant biotrophs that may contain endobacteria in their cytoplasm. Genome sequencing of Candidatus Glomeribacter gigasporarum revealed a reduced genome and dependence on the fungal host. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of the endobacterium indicated that endobacteria have an important role in the fungal pre-symbiotic phase by enhancing fungal bioenergetic capacity. To improve the understanding of fungal-endobacterial interactions, iTRAQ (isobaric tags for relative and absolute quantification) quantitative proteomics was used to identify differentially expressed proteins in G. margarita germinating spores with endobacteria (B+), without endobacteria in the cured line (B-) and after application of the synthetic strigolactone GR24. Proteomic, transcriptomic and biochemical data identified several fungal and bacterial proteins involved in interspecies interactions. Endobacteria influenced fungal growth, calcium signalling and metabolism. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than in B-. A shift towards pentose phosphate metabolism was detected in B-. Quantification of carbonylated proteins indicated that the B- line had higher oxidative stress levels, which were also observed in two host plants. This study shows that endobacteria generate a complex interdomain network that affects AMF and fungal-plant interactions.

Cyclic AMP deficiency negatively affects cell growth and enhances stress-related responses in tobacco Bright Yellow-2 cells.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a recognized second messenger; however, knowledge of cAMP involvement in plant physiological processes originates primarily from pharmacological studies. To obtain direct evidence for cAMP function in plants, tobacco Bright Yellow-2 (BY-2) cells were transformed with the cAMP sponge, which is a genetically encoded tool that reduces cAMP availability. BY-2 cells expressing the cAMP sponge (cAS cells), showed low levels of free cAMP and exhibited growth inhibition that was not proportional to the cAMP sponge transcript level. Growth inhibition in cAS cells was closely related to the precocious inhibition of mitosis due to a delay in cell cycle progression. The cAMP deficiency also enhanced antioxidant systems. Remarkable changes occurred in the cAS proteomic profile compared with that of wild-type (WT) cells. Proteins involved in translation, cytoskeletal organization, and cell proliferation were down-regulated, whereas stress-related proteins were up-regulated in cAS cells. These results support the hypothesis that BY-2 cells sense cAMP deficiency as a stress condition. Finally, many proteasome subunits were differentially expressed in cAS cells compared with WT cells, indicating that cAMP signaling broadly affects protein degradation via the ubiquitin/proteasome pathway.

Chemical, molecular, and proteomic analyses of moss bag biomonitoring in a petrochemical area of Sardinia (Italy).

In this study, Hypnum cupressiforme moss bags were used to examine the atmospheric deposition of trace elements in the oil refinery region of Sardinia (Italy) compared with surrounding natural zones. The concentrations of 13 elements [arsenic (As), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn)] were determined using inductively coupled plasma optical emission spectrometry. A significant accumulation of pollutants was detected using active biomonitoring with moss bags compared with a control site. The most relevant contaminants for all of the tested sites were Cr, Cu, Ni, and Zn. Moreover, the accumulation of Cr and Zn in the refinery industrial areas, IA1 and IA2, was more than five times greater than that detected at the control site. Levels of Cd, Mg, and Pb were also higher at all of the monitored sites compared with the control site. Both genomic and proteomic methods were used to study the response of H. cupressiforme to air pollution. No DNA damage or mutations were detected using the amplified fragment length polymorphisms (AFLP) method. At the protein level, 15 gel spots exhibited differential expression profiles between the moss samples collected at the IA1 site and the control site. Furthermore, among the 14 spots that showed a decrease in protein expression, nine were associated with ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and proteins of the light-harvesting complexes of photosystem (PS) II, three were associated with protein synthesis, and three were stress-related proteins. Thus, some of these proteins may represent good moss biosensors which could be used as pre-alert markers of environmental pollution.

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

BACKGROUND: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. METHODS: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. RESULTS: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. CONCLUSIONS: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

Uptake and effects of a mixture of widely used therapeutic drugs in Eruca sativa L. and Zea mays L. plants.

Pharmaceutically active compounds (PACs) are continuously dispersed into the environment due to human and veterinary use, giving rise to their potential accumulation in edible plants. In this study, Eruca sativa L. and Zea mays L. were selected to determine the potential uptake and accumulation of eight different PACs (Salbutamol, Atenolol, Lincomycin, Cyclophosphamide, Carbamazepine, Bezafibrate, Ofloxacin and Ranitidine) designed for human use. To mimic environmental conditions, the plants were grown in pots and irrigated with water spiked with a mixture of PACs at concentrations found in Italian wastewaters and rivers. Moreover, 10× and 100× concentrations of these pharmaceuticals were also tested. The presence of the pharmaceuticals was tested in the edible parts of the plants, namely leaves for E. sativa and grains for Z. mays. Quantification was performed by liquid chromatography mass spectroscopy (LC/MS/MS). In the grains of 100× treated Z. mays, only atenolol, lincomycin and carbamazepine were above the limit of detection (LOD). At the same concentration in E. sativa plants the uptake of all PACs was >LOD. Lincomycin and oflaxacin were above the limit of quantitation in all conditions tested in E. sativa. The results suggest that uptake of some pharmaceuticals from the soil may indeed be a potential transport route to plants and that these environmental pollutants can reach different edible parts of the selected crops. Measurements of the concentrations of these pharmaceuticals in plant materials were used to model potential adult human exposure to these compounds. The results indicate that under the current experimental conditions, crops exposed to the selected pharmaceutical mixture would not have any negative effects on human health. Moreover, no significant differences in the growth of E. sativa or Z. mays plants irrigated with PAC-spiked vs. non-spiked water were observed.

Phytotoxic and genotoxic effects of silver nanoparticles exposure on germinating wheat seedlings.

We investigated the effects of 1 and 10 mg L(-1) AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L(-1) AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs. To gain an increased understanding of the molecular response to AgNP exposure, we analyzed the genomic and proteomic changes induced by AgNPs in wheat seedlings. At the DNA level, we applied the AFLP technique and we found that both treatments did not induce any significant DNA polymorphisms. 2DE profiling of roots and shoots treated with 10 mg L(-1) of AgNPs revealed an altered expression of several proteins mainly involved in primary metabolism and cell defense.