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.

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.

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.

GUN1 influences the accumulation of NEP-dependent transcripts and chloroplast protein import in Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis.

Correct chloroplast development and function require co-ordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast-localized pentatricopeptide repeat (PPR) protein with a C-terminal Small MutS-Related (SMR) domain, is involved in integrating multiple developmental and stress-related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signalling pathways. Here we show that following perturbation of chloroplast protein homeostasis: (i) by growth in lincomycin-containing medium; or (ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21-1 and prpl11-1) or plastid protein import and folding (cphsc70-1), GUN1 influences NEP-dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.

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.

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.

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.

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.

The soluble proteome of tobacco Bright Yellow-2 cells undergoing H2O2-induced programmed cell death.

Plant programmed cell death (PCD) is a genetically controlled process that plays an important role in development and stress responses. Reactive oxygen species (ROS) are key inducers of PCD. The addition of 50 mM H2O2 to tobacco Bright Yellow-2 (TBY-2) cell cultures induces PCD. A comparative proteomic analysis of TBY-2 cells treated with 50 mM H2O2 for 30 min and 3 h was performed. The results showed early down-regulation of several elements in the cellular redox hub and inhibition of the protein repair-degradation system. The expression patterns of proteins involved in the homeostatic response, in particular those associated with metabolism, were consistently altered. The changes in abundance of several cytoskeleton proteins confirmed the active role of the cytoskeleton in PCD signalling. Cells undergoing H2O2-induced PCD fail to cope with oxidative stress. The antioxidant defence system and the anti-PCD signalling cascades are inhibited. This promotes a genetically programmed cell suicide pathway. Fifteen differentially expressed proteins showed an expression pattern similar to that previously observed in TBY-2 cells undergoing heat shock-induced PCD. The possibility that these proteins are part of a core complex required for PCD induction is discussed.

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.

Effects of a complex mixture of therapeutic drugs on unicellular algae Pseudokirchneriella subcapitata.

Pharmaceutically-active compounds are regularly and widely released into the aquatic environment in an unaltered form or as metabolites. So far, little is known about their potential detrimental effects on algae populations which can ultimately impact nutrient cycling and oxygen balance. For our analysis, the common microalga Pseudokirchneriella subcapitata (P. subcapitata) was exposed to a mixture of 13 drugs found in Italian wastewaters and rivers. Traces of pharmaceuticals investigated were detected in treated algal cells, except for cyclophosphamide and ranitidine, indicating that these algae are able to absorb pharmaceutical pollutants from the environment. The effects of the treatment were investigated by Amplified Fragment Length Polymorphism (AFLP) assessment of DNA damage and 2-DE proteomic analysis. While no genotoxic effect was detected, proteomic analysis showed that algae are sensitive to the presence of drugs and that, in particular, the chloroplast is affected.