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.

Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling.

The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insights into cellular responses to impaired plastid protein homeostasis.

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.

The Role of Protein-Rich Extracts from Chondrus crispus as Biostimulant and in Enhancing Tolerance to Drought Stress in Tomato Plants.

The application of seaweed extract-based biostimulants is a promising approach for achieving sustainable agriculture, with an enormous potential of improving crop yield and mitigating climate change effects. Abiotic stressors, such as drought, are major factors resulting in tomato (Solanum lycopersicum L.) yield losses and seaweed-based biostimulants have been proposed as an eco-friendly strategy to counteract this negative impact. Chondrus crispus is a common red seaweed widely used as source of carrageenans, not yet explored as a plant biostimulant. In this study, a protein hydrolysate-rich C. crispus extract, by-products of the carrageenan extraction, was tested on tomato plants under well-watered condition and water shortage. The foliar application of the protein-rich C. crispus extract conferred drought tolerance to tomato plants resulting in less noticeable visual stress symptoms. Treated plants showed higher shoot height and biomass under both well-watered and water deficit conditions, evidencing the double effect exerted by this new biostimulant, as plant growth promoter and drought stress protector. The treatment with the biostimulant had an effect on levels of abscisic acid and proline, and triggered the expression of Solyc02g084840, a drought marker gene. Finally, a label-free mass spectrometric approach allowed us to identify phycoerythrins and phycocyanins as major bioactive proteins contained in the extract. Altogether, these results indicate that the foliar application of protein hydrolysate-rich extracts from C. crispus improved tomato plant growth and tolerance to drought stress, suggesting a new opportunity for further applications in the agriculture and horticultural sectors.

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.

Efficient extraction of carrageenans from Chondrus crispus for the green synthesis of gold nanoparticles and formulation of printable hydrogels.

The integral utilization of sustainable resources with versatile, efficient and cleaner processes is encouraged. Hydrothermal treatment with subcritical water is a chemical free, tunable and rapid technology providing enhanced yield compared to conventional extraction and was explored for the benign by design extraction and depolymerization of carrageenan from Chondrus crispus. Up to 90% of the seaweed was solubilized operating under nonisothermal regime during heating up to 200 °C and 75.5% crude carrageenan yield was attained at 140 °C. Crude carrageenan could not be precipitated by ethanol from the extracts produced at 180 °C and higher temperatures, but ultrafiltration (100 kDa) of the extract obtained at 160 °C provided comparable recovery yields and similar rheological features to those of the ethanol precipitated product. Operation at 140 °C was preferred based on the higher recovery yield of the biopolymer and the whole extract was suitable for the green synthesis of polycrystalline decahedral quasi-spherical gold nanoparticles with a mean size distribution of 8.4 nm and Z potential value of -40.2 mV. Alternatively, the crude carrageen fraction was used for the formulation of printable biopolymer based gels with suitable mechanical properties, including a relevant gel strength enhancement (about 10-fold) when compared with conventional procedures.

Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

N-Terminally Truncated and Pyroglutamate-Modified Aß Forms Are Measurable in Human Cerebrospinal Fluid and Are Potential Markers of Disease Progression in Alzheimer's Disease.

Alzheimer's disease (AD) is a pathology characterized by the accumulation in the brain of intracellular and extracellular amyloid-ß (Aß) aggregates, especially of Aß1-40 and Aß1-42 peptides. It is known that N-terminally truncated or modified Aß forms also exist in AD brains and cerebrospinal fluid (CSF), and they play a key role in the pathogenesis of the disease. Herein, we developed an antibody-free method based on Solid-Phase Extraction and Electrospray Ionization Liquid Chromatography Mass Spectrometry for the identification and quantitation in human CSF of Aß isoforms. In human CSF, we could detect and quantify a panel of 19 Aß isoforms, including N-terminally truncated and pyroglutamate-modified forms, never quantified before in CSF. Among these, we identified novel N-terminally truncated Aß species: four bound to copper and two phosphorylated forms, which were found to be the most common proteoforms in human CSF along with Aß1-40, Aß3-40, and AßpE11-42. We tested the newly developed and validated method in a pilot study on CSF from elderly individuals with subjective memory complaints (SMCs, n = 9), mild cognitive impairment (MCI, n = 18), and AD (n = 15); along with Aß1-42, five N-terminally truncated forms (Aß11-40, Aß3-42, AßpE11-42, AßpE3-40, and Aß4-40 Cu2+) are altered in AD/MCI. Thus, we demonstrated that N-terminally truncated and pyroglutamate-modified Aß can be quantified in human CSF, and five of them, along with Aß1-42, are potential markers of AD progression. The described method could represent a useful tool for patients' stratification and monitoring. Moreover, the newly identified Aß CSF species might represent new potential therapeutic targets.

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.

Cyclic AMP mediates heat stress response by the control of redox homeostasis and ubiquitin-proteasome system.

Heat stress (HS), causing impairment in several physiological processes, is one of the most damaging environmental cues for plants. To counteract the harmful effects of high temperatures, plants activate complex signalling networks, indicated as HS response (HSR). Expression of heat shock proteins (HSPs) and adjustment of redox homeostasis are crucial events of HSR, required for thermotolerance. By pharmacological approaches, the involvement of cAMP in triggering plant HSR has been recently proposed. In this study, to investigate the role of cAMP in HSR signalling, tobacco BY-2 cells overexpressing the 'cAMP-sponge', a genetic tool that reduces intracellular cAMP levels, have been used. in vivo cAMP dampening increased HS susceptibility in a HSPs-independent way. The failure in cAMP elevation during HS caused a high accumulation of reactive oxygen species, due to increased levels of respiratory burst oxidase homolog D, decreased activities of catalase and ascorbate peroxidase, as well as down-accumulation of proteins involved in the control of redox homeostasis. In addition, cAMP deficiency impaired proteasome activity and prevented the accumulation of many proteins of ubiquitin-proteasome system (UPS). By a large-scale proteomic approach together with in silico analyses, these UPS proteins were identified in a specific cAMP-dependent network of HSR.

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.

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.

Morphological and proteomic responses of Eruca sativa exposed to silver nanoparticles or silver nitrate.

Silver nanoparticles (AgNPs) are widely used in commercial products, and there are growing concerns about their impact on the environment. Information about the molecular interaction of AgNPs with plants is lacking. To increase our understanding of the mechanisms involved in plant responses to AgNPs and to differentiate between particle specific and ionic silver effects we determined the morphological and proteomic changes induced in Eruca sativa (commonly called rocket) in response to AgNPs or AgNO3. Seedlings were treated for 5 days with different concentrations of AgNPs or AgNO3. A similar increase in root elongation was observed when seedlings were exposed to 10 mg Ag L(1) of either PVP-AgNPs or AgNO3. At this concentration we performed electron microscopy investigations and 2-dimensional electrophoresis (2DE) proteomic profiling. The low level of overlap of differentially expressed proteins indicates that AgNPs and AgNO3 cause different plant responses. Both Ag treatments cause changes in proteins involved in the redox regulation and in the sulfur metabolism. These responses could play an important role to maintain cellular homeostasis. Only the AgNP exposure cause the alteration of some proteins related to the endoplasmic reticulum and vacuole indicating these two organelles as targets of the AgNPs action. These data add further evidences that the effects of AgNPs are not simply due to the release of Ag ions.

Physiological and molecular effects associated with palladium treatment in Pseudokirchneriella subcapitata.

Human activities have increased the levels of environmental palladium (Pd) worldwide. Due to the growing evidence of its toxicity, Pd pollution has become the focus of serious concern. Several studies have given an account of the increasing concentration of Pd in aquatic ecosystems. The aim of the current study is to analyze the physiological and molecular effects induced by Pd on freshwater unicellular green algae. To do this, Pseudokirchneriella subcapitata (P. subcapitata) was exposed in vitro to different concentrations (0.1, 0.25 and 0.5 mg l(-1)) of K(2)PdCl(4), a soluble salt of Pd, corresponding to 0.03, 0.075 and 0.15 mg l(-1) of Pd. The uptake and the effects on algal growth and morphology were determined. The main results are that Pd is able to induce damage in P. subcapitata at a concentration of 0.1 mg l(-1) of K(2)PdCl(4), with the damage becoming more evident at a concentration of 0.25 mg l(-1)of K(2)PdCl(4); at a concentration of 0.5 mg l(-1) of K(2)PdCl(4), cellular degeneration occurs. The main cellular target of Pd is the chloroplast, as shown by TEM and proteomic analysis. TEM analysis also showed accumulation of precipitates, probably of Pd, in the chloroplasts, although further experiments are necessary to confirm that these are Pd-precipitates. Amplified fragment length polymorphism analysis (AFLP) demonstrated that Pd, even at the lowest concentration tested, induced randomly distributed DNA changes either directly or indirectly in the algal genome and that oxidative processes were involved.