Uptake and distribution of perfluoroalkyl substances by grafted tomato plants cultivated in a contaminated site in northern Italy.

Poly- and perfluoroalkyl substances (PFAS) are highly persistent and mobile pollutants raising alarming concerns due to their capability to accumulate in living organisms and exert toxic effects on human health. We studied the accumulation of different PFAS in the leaves and fruits of tomato plants grown on a PFAS-polluted soil in North-East Italy. Tomato plants were grafted with different rootstocks characterized by different vigor, and irrigated with PFAS-polluted groundwater. Leaves and fruits of the first and sixth truss were analyzed at full plant maturity. All tomato varieties accumulated PFAS in leaves and fruits, with the highest concentrations detected in the most vigorous rootstock and reflecting the PFAS concentration profile of the irrigation water. PFAS with a chain length from 4 to 8 C atoms and with carboxylic and sulfonic functional groups were detected in plant leaves, whereas only carboxylic C4, C5, and C6 PFAS were detected in tomato fruits. A general trend of decreasing PFAS concentrations in fruits upon increasing height of the plant trusses was observed. Calculation of the target hazard quotient (THQ) showed increasing values depending on the plant vigor. The hazard index (HI) values showed values slightly higher than 1 for the most vigorous plants, indicating potential risks to human health associated with the consumption of contaminated tomato fruits.

Perfluoroalkyl substances exposure alters stomatal opening and xylem hydraulics in willow plants.

Climate change and pollution are increasingly important stress factors for life on Earth. Dispersal of poly- and perfluoroalkyl substances (PFAS) are causing worldwide contamination of soils and water tables. PFAS are partially hydrophobic and can easily bioaccumulate in living organisms, causing metabolic alterations. Different plant species can uptake large amounts of PFAS, but little is known about its consequences for the plant water relation and other physiological processes, especially in woody plants. In this study, we investigated the fractionation of PFAS bioaccumulation from roots to leaves and its effects on the conductive elements of willow plants. Additionally, we focused on the stomal opening and the phytohormonal content. For this purpose, willow cuttings were exposed to a mixture of 11 PFAS compounds and the uptake was evaluated by LC-MS/MS. Stomatal conductance was measured and the xylem vulnerability to air embolism was tested and further, the abscisic acid and salicylic acid contents were quantified using LC-MS/MS. PFAS accumulated from roots to leaves based on their chemical structure. PFAS-exposed plants showed reduced stomatal conductance, while no differences were observed in abscisic acid and salicylic acid contents. Interestingly, PFAS exposure caused a higher vulnerability to drought-induced xylem embolism in treated plants. Our study provides novel information about the PFAS effects on the xylem hydraulics, suggesting that the plant water balance may be affected by PFAS exposure. In this perspective, drought events may be more stressful for PFAS-exposed plants, thus reducing their potential for phytoremediation.

Perfluorinated alkyl substances affect the growth, physiology and root proteome of hydroponically grown maize plants.

Poly- and perfluorinated alkyl substances (PFAS) are a group of persistent organic pollutants causing serious global concern. Plants can accumulate PFAS but their effect on plant physiology, especially at the molecular level is not very well understood. Hence, we used hydroponically-grown maize plants treated with a combination of eleven different PFAS (each at 100 µg L-1) to investigate their bioaccumulation and effects on the growth, physiology and their impact on the root proteome. A dose-dependent decrease in root growth parameters was evidenced with a significant reduction in the relative growth rate, fresh weight of leaves and roots and altered photosynthetic parameters in PFAS-treated plants. Higher concentration of shorter PFAS (C < 8) was detected in the leaves, while long-chain PFAS (C ≥ 8) were more retained in roots. From the root proteome analysis, we identified 75 differentially abundant proteins, mostly involved in cellular metabolic and biosynthetic processes, translation and cytoskeletal reorganization. Validating the altered protein abundance using quantitative real-time PCR, the results were further substantiated using amino acid and fatty acid profiling, thus, providing first insight into the altered metabolic state of plants exposed to PFAS from a proteomics perspective.

Quantitative Proteomics of Maize Roots Treated with a Protein Hydrolysate: A Comparative Study with Transcriptomics Highlights the Molecular Mechanisms Responsive to Biostimulants.

Protein hydrolysate (PH)-based biostimulants offer a cost-effective and sustainable approach for the regulation of physiological processes in plants to stimulate growth and improve stress tolerance. Understanding the mode of action of PHs is challenging, but it is indispensable to improve existing candidates and to develop novel molecules with enhanced stimulatory effects. Hence, the present study aimed to understand the proteome level responses in the B73 maize roots treated with APR, a PH biostimulant, at two increasing concentrations and to compare and integrate it with the transcriptomic data obtained previously under identical experimental conditions. Results indicate that APR induced dose-dependent global changes in the transcriptome and proteome of maize roots. APR treatment altered the expression and abundance of several genes and proteins related to redox homeostasis, stress response, glycolysis, tricarboxylic acid cycle, pentose phosphate pathway, and other metabolic pathways of carbohydrates, amino acids, and lipids. Further, metabolic processes of phytohormone, secondary metabolites, especially phenylpropanoids, flavonoids, and terpenoids and transport, and cytoskeletal reorganization associated mechanisms were stimulated. Our results suggest that APR treatment altered the redox homeostasis and thus triggered an oxidative signal. This could be one of the key regulators of the cascade of downstream events involving multiple signaling, hormonal, and metabolic pathways, resulting in an altered physiological and metabolic state which consequently could lead to improved growth and stress adaptation observed in biostimulant-treated plants.

Accumulation and effects of perfluoroalkyl substances in three hydroponically grown Salix L. species.

The potential of young rooted cuttings of three Salix L. species plants to accumulate a mixture of eleven perfluoroalkyl substances (PFASs), in particular, perfluoroalkyl acids (PFAAs), from the nutrient solution and their effects on plant growth and photosynthesis were assessed in an 8-day experiment. The growth rate of the willow plants exposed to the PFAA mixture was not much affected except for S. triandra. Regarding photosynthesis, the gas exchange parameters were affected more than those related to chlorophyll fluorescence, with significant increase of the net CO2 assimilation rate and parameters related to stomatal conductance. A decreasing trend in the PFAA concentration in leaves with increasing carbon chain length was observed, whereas long-chain PFAAs showed higher concentrations in roots. Accordingly, the foliage to root concentration factor highlighted that PFAAs with shorter carbon chain length (C ≤ 7) translocated and accumulated relatively more in leaves compared to roots. Removal efficiency of individual PFAAs for leaves and roots were comparatively higher with S. eleagnos and S. purpurea than S. triandra, with mean removal values at the whole plant level ranging around 10% of the amount initially spiked, suggesting their potential for phytoremediation of PFASs.

A proteomic and biochemical investigation on the effects of sulfadiazine in Arabidopsis thaliana.

Animal manure or bio-solids used as fertilizers are the main routes of antibiotic exposure in the agricultural land, which can have immense detrimental effects on plants. Sulfadiazine (SDZ), belonging to the class of sulfonamides, is one of the most detected antibiotics in the agricultural soil. In this study, the effect of SDZ on the growth, changes in antioxidant metabolite content and enzyme activities related to oxidative stress were analysed. Moreover, the proteome alterations in Arabidopsis thaliana roots in response to SDZ was examined by means of a combined iTRAQ-LC-MS/MS quantitative proteomics approach. A dose-dependent decrease in leaf biomass and root length was evidenced in response to SDZ. Increased malondialdehyde content at higher concentration (2 µM) of SDZ indicated increased lipid peroxidation and suggest the induction of oxidative stress. Glutathione levels were significantly higher compared to control, whereas there was no increase in ascorbate content or the enzyme activities of glutathione metabolism, even at higher concentrations. In total, 48 differentially abundant proteins related to stress/stimuli response followed by transcription and translation, metabolism, transport and other functions were identified. Several proteins related to oxidative, dehydration, salinity and heavy metal stresses were represented. Upregulation of peroxidases was validated with total peroxidase activity. Pathway analysis provided an indication of increased phenylpropanoid biosynthesis. Probable molecular mechanisms altered in response to SDZ are highlighted.

Apoplastic gamma-glutamyl transferase activity encoded by GGT1 and GGT2 is important for vegetative and generative development.

Gamma-glutamyl transferase (GGT; EC 2.3.2.2) is the only enzyme capable of degrading glutathione (GSH) in extra-cytosolic spaces. In plant cells, the GGT1 and GGT2 isoforms are located in the apoplast, bound respectively to the cell wall and the plasma membrane. GGT1 is expressed throughout plants, mainly in the leaves and vascular system, while GGT2 is more specifically expressed in seeds and trichomes, and weakly in roots. Their role in plant physiology remains to be clarified, however. Obtaining the ggt1/ggt2 double mutant can offer more clues than the corresponding single mutants, and to prevent any compensatory expression between the two isoforms. In this work, ggt1/ggt2 RNAi (RNA interference) lines were generated and characterized in the tissues where both isoforms are expressed. The seed yield was lower in the ggt1/ggt2 RNAi plants due to the siliques being fewer in number and shorter in length, with no changes in thiols and sulfur compounds. Proline accumulation and delayed seed germination were seen in one line. There were also fewer trichomes (which contain high levels of GSH) in the RNAi lines than in the wild type, and the root elongation rate was slower. In conclusion, apoplastic GGT silencing induces a decrease in the number of organs with a high GSH demand (seeds and trichomes) as a result of resource reallocation to preserve integrity and composition.

In vitro secretomic analysis identifies putative pathogenicity-related proteins of Sporisorium scitamineum - The sugarcane smut fungus.

Sporisorium scitamineum, the sugarcane smut pathogen, relies predominantly on its secretome to successfully colonise its host, in accordance with other related smut fungi. Considering the significance of deciphering its secretome, we have examined alterations in the in vitro secretome of S. scitamineum in response to synthetic and sugarcane meristem tissue-amended growth media, so as to identify host signal responsive secretory proteins. Secretory proteins that were differentially abundant and exclusively secreted in response to host extract media were identified by two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF MS. Of the 16 differentially abundant and exclusively secreted proteins, nine proteins were identified. Among which, six were related to cell wall modification, morphogenesis, polysaccharide degradation, and carbohydrate metabolism. In planta gene expression profiling indicated that five in vitro secreted proteins were expressed in distinct patterns by S. scitamineum during different stages of infection with relatively higher expression at 1 day after inoculation, suggesting that these proteins could be aiding S. scitamineum at early time points in penetration and colonisation of sugarcane cells. The present study has provided insights into the alterations occurring in the secretome of S. scitamineum at in vitro conditions and has resulted in the identification of secretory proteins that are possibly associated with pathogenicity of the sugarcane smut fungus.

Proteomic analysis of a compatible interaction between sugarcane and Sporisorium scitamineum.

Smut caused by Sporisorium scitamineum is one of the important diseases of sugarcane with global significance. Despite the intriguing nature of sugarcane, S. scitamineum interaction, several pertinent aspects remain unexplored. This study investigates the proteome level alterations occurring in the meristem of a S. scitamineum infected susceptible sugarcane cultivar at whip emergence stage. Differentially abundant proteins were identified by 2DE coupled with MALDI-TOF/TOF-MS. Comprehensively, 53 sugarcane proteins identified were related to defence, stress, metabolism, protein folding, energy, and cell division; in addition, a putative effector of S. scitamineum, chorismate mutase, was identified. Transcript expression vis-à-vis the activity of phenylalanine ammonia lyase was relatively higher in the infected meristem. Abundance of seven candidate proteins in 2D gel profiles was in correlation with its corresponding transcript expression levels as validated by qRT-PCR. Furthermore, this study has opened up new perspectives on the interaction between sugarcane and S. scitamineum.

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation.

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for Reactive Oxygen Species (ROS) generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform (GGT1) bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with Ultraviolet-B radiation (UV-B) and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of Hydrogen Peroxide (H2O2), which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807.

A note on protein expression changes in chicken breast muscle in response to time in transit before slaughtering.

Aims of the research were to devise a proteome map of the chicken Pectoralis superficialis muscle, as resolved by two-dimensional gel electrophoresis, and to characterize protein expression changes in the soluble protein fraction in commercial conditions due to age and to time in transit before slaughtering. Broilers were reared under commercial conditions until they reached a mean 1.8 kg and 36 d, or 2.6 kg and 46 d of age. Transport to the slaughterhouse took 90 or 220 minutes. Transport-induced stress was assessed from blood metabolites and leukocyte cell counts, revealing significant changes in albumin, glucose and triglyceride concentrations, in heterophils and leukocyte counts for chickens in transit for longer, and in glucose depending mainly on age. The sarcoplasmic protein fractions were extracted from a total of 39 breast muscle samples, collected 15 min post mortem, for analysis by two-dimensional electrophoresis. Image and statistical analyses enabled us to study the qualitative and quantitative differences between the samples. Twelve up- or down-regulated protein spots were detected (P < 0.05): 8 related to the age effect, 2 to time in transit, and 2 to the interaction between the two. Age and time in transit influenced the avian proteome regulating the biological processes linked to the cellular housekeeping functions, related mainly to metabolism, cell division and control of apoptosis. Principal component analysis clustering was used to assess differences between birds. Age difference discriminated between the chickens analyzed better than time in transit, which seemed to have less general impact on the proteome fraction considered here. Isolating and identifying the proteins whose expression changes in response to transport duration and age shed some light on the biological mechanisms underlying growth and stress-related metabolism in chickens. Our results, combined with a further characterization of the chicken proteome associated with commercial chicken slaughtering management, will hopefully inspire alternative strategies and policies, and action to reduce the impact of stress related to time in transit.

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma-glutamyl cycle in plant's adaptation to environment.

The existence of a gamma-glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma-glutamyl transferase (GGT)-assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild-type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low-molecular weight range compared to WT. The combined iTRAQ and LC-MS/MS-based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH-associated genes showed that disruption of gamma-glutamyl cycle in ggt1 knockout-leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma-glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana.

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 µM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.

Carbon and nitrogen metabolism in barley plants exposed to UV-B radiation.

The effect of UV-B radiation on FW, leaf and stem length, photosynthetic O2 evolution, levels of carbohydrates and nitrates, and extractable activities of some of the enzymes involved in C and N metabolism was evaluated in barley (Hordeum vulgare L. cv. Express) seedlings during the 9 days following transfer to an UV-B enriched environment. The results show that under our experimental conditions UV-B radiation scarcely affects the photosynthetic competence of barley leaves, expressed as RuBP carboxylase (EC 4.1.1.39) activity, O2 evolution rate and chlorophyll content. Nevertheless, this treatment induced significant alterations of the enzyme activity of nitrate reductase (EC 1.6.6.1) and glutamine synthetase (EC 6.3.1.2), although only after a few days of treatment. The effects were not confined to the exposed tissue, but were detectable also at the root level. In fact, nitrate reductase decreased in response to UV-B in both leaf and root tissue, whereas glutamine synthetase was affected only in the root. In contrast, nitrate content was not influenced by the treatment, neither in root nor in leaf tissue, whilst leaf sucrose diminished in exposed plants only on the last day of treatment.