Subchronic oral exposure to polystyrene microplastics affects hepatic lipid metabolism, inflammation, and oxidative balance in gilthead seabream (Sparus aurata).

Microplastics (MPs) pose a clear threat to aquatic organisms affecting their health. Their impact on liver homeostasis, as well as on the potential onset of nonalcoholic fatty liver disease (NAFLD), is still poorly investigated and remains almost unknown. The aim of this study was to evaluate the outcomes of subchronic exposure to polystyrene MPs (PS-MPs; 1-20 µm; 0, 25, or 250 mg/kg b.w./day) on lipid metabolism, inflammation, and oxidative balance in the liver of gilthead seabreams (Sparus aurata Linnaeus, 1758) exposed for 21 days via contaminated food. PS-MPs induced an up-regulation of mRNA levels of crucial genes associated with lipid synthesis and storage (i.e., PPARy, Srebp1, Fasn) without modifications of genes involved in lipid catabolism (i.e., PPARα, HL, Pla2) or transport and metabolism (Fabp1) in the liver. The increase of CSF1R and pro-inflammatory cytokines gene expression (i.e., TNF-α and IL-1ß) was also observed in exposed fish in a dose-dependent manner. These findings were confirmed by hepatic histological evaluations reporting evidence of lipid accumulation, inflammation, and necrosis. Moreover, PS-MPs caused the impairment of the hepatic antioxidant defense system through the alteration of its enzymatic (catalase, superoxide dismutase, and glutathione reductase) and non-enzymatic (glutathione) components, resulting in the increased production of reactive oxygen species (ROS) and malondialdehyde (MDA), as biomarkers of oxidative damage. The alteration of detoxifying enzymes was inferred by the decreased Ethoxyresorufin-O-deethylase (EROD) activity and the increased activity of glutathione-S-transferase (GST) at the highest PS-MP dose. The study suggests that PS-MPs affect the liver health of gilthead seabream. The liver dysfunction and damage caused by exposure to PS-MPs result from a detrimental interplay of inflammation, oxidative damage, and antioxidant and detoxifying enzymatic systems modifications, altering the gut-liver axis homeostasis. This scenario is suggestive of the involvement of MP-induced effects in the onset and progression of hepatic lipid dysfunction in gilthead seabream.

Biological responses to heavy metal stress in the moss Leptodictyum riparium (Hedw.) Warnst.

Leptodictyum riparium, a widely distributed aquatic moss, can both tolerate and accumulate very high concentrations of toxic heavy metals, with only slight apparent damage. Here we report the effects on photosynthetic yield, glutathione (GSH), phytochelatin (PCn) synthesis, nitrogen metabolism and cellular localization of molecules rich in SH groups in L. riparium exposed in vitro to heavy metals. We simulated the concentrations of Cu, Zn, Cd, Pb detected in Regi Lagni, Italy, one of the most contaminated freshwater sites in Southern Europe, in the laboratory to test how the moss responds to heavy metal contamination. There was a steady decrease of photosynthetic efficiency correlated with the heavy metal concentrations and ultrastructural organization. All PCn levels increased significantly as the concentration of heavy metals increased, while the GSH levels did not appear to be particularly affected. A significant increase of GDH and NADH-GOGAT activities increased with increasing heavy metal concentration. Immunoblotting analysis revealed an increase of the chl-GS2 while no significant increase was detected in the cyt-GS1. These results give insight into the molecular events underlying the metal-tolerance of the aquatic moss L. riparium exposed to environmental heavy metal concentrations.

Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7.

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF.

Bioinformatic Characterization of Sulfotransferase Provides New Insights for the Exploitation of Sulfated Polysaccharides in Caulerpa.

Caulerpa is an unusual algal genus from Caulerpaceae (Chlorophyta, Bryopsidales). Species from this family produce a wide range of metabolites suitable for biotechnology applications. Among these, sulfated polysaccharides (SPs) are often highly desirable for pharmaceutical and nutraceutical applications. Here, we provide a classification of sulfotransferases from Caulerpa; these important enzymes catalyze the nodal step for the biosynthesis of SPs. For this, we performed phylogenetic, genomic, expression analyses and prediction of the protein structure on sulfotransferases from Caulerpa. Sequences, domains and structures of sulfotransferases generally shared common characteristics with other plants and algae. However, we found an extensive duplication of sulfotransferase gene family, which is unique among the green algae. Expression analysis revealed specific transcript abundance in the pinnae and rachis of the alga. The unique genomic features could be utilized for the production of complex SPs, which require multiple and specific sulfation reactions. The expansion of this gene family in Caulerpaceae would have resulted in a number of proteins characterizing the unique SPs found in these algae. We provide a putative biosynthetic pathway of SPs, indicating the unique characteristics of this pathway in Caulerpa species. These data may help in the future selection of Caulerpa species for both commercial applications and genetic studies to improve the synthesis of valuable products from Caulerpa.

Impact of Nitrogen Nutrition on Cannabis sativa: An Update on the Current Knowledge and Future Prospects.

Nitrogen (N) availability represents one of the most critical factors affecting cultivated crops. N is indeed a crucial macronutrient influencing major aspects, from plant development to productivity and final yield of lignocellulosic biomass, as well as content of bioactive molecules. N metabolism is fundamental as it is at the crossroad between primary and secondary metabolic pathways: Besides affecting the synthesis of fundamental macromolecules, such as nucleic acids and proteins, N is needed for other types of molecules intervening in the response to exogenous stresses, e.g. alkaloids and glucosinolates. By partaking in the synthesis of phenylalanine, N also directly impacts a central plant metabolic 'hub'-the phenylpropanoid pathway-from which important classes of molecules are formed, notably monolignols, flavonoids and other types of polyphenols. In this review, an updated analysis is provided on the impact that N has on the multipurpose crop hemp (Cannabis sativa L.) due to its renewed interest as a multipurpose crop able to satisfy the needs of a bioeconomy. The hemp stalk provides both woody and cellulosic fibers used in construction and for biocomposites; different organs (leaves/flowers/roots) are sources of added-value secondary metabolites, namely cannabinoids, terpenes, flavonoids, and lignanamides. We survey the available literature data on the impact of N in hemp and highlight the importance of studying those genes responding to both N nutrition and abiotic stresses. Available hemp transcriptomic datasets obtained on plants subjected to salt and drought are here analyzed using Gene Ontology (GO) categories related to N metabolism. The ultimate goal is to shed light on interesting candidate genes that can be further studied in hemp varieties growing under different N feeding conditions and showing high biomass yield and secondary metabolite production, even under salinity and drought.

Zn-biofortification enhanced nitrogen metabolism and photorespiration process in green leafy vegetable Lactuca sativa L.

BACKGROUND: Excessive rates of nitrogen (N) fertilizers may result in elevated concentrations of nitrate (NO3- ) in plants. Considering that many programs of biofortification with trace elements are being performed, it has become important to study how the application of these elements affects plant physiology and, particularly, N utilization in leaf crops. The main objective of the present study was to determine whether the NO3- accumulation and the nitrogen use efficiency was affected by the application of different doses of Zn in Lactuca sativa plants. RESULTS: Zn doses in the range 80-100 µmol L-1 produced an increase in Zn concentration provoking a decrease of NO3- concentration and increase of the nitrate reductase, glutamine synthetase and aspartate aminotransferase activities, as well as the photorespiration processes. As result, we observed an increase in reduced N, total N concentration and N utilization efficiency. Consequently, at a dose of 80 µmol L-1 of Zn, the amino acid concentration increased significantly. CONCLUSION: Adequate Zn fertilization is an important critical player in lettuce, especially at a dose of 80 µmol L-1 of Zn, because it could result in an increase in the Zn concentration, a reduction of NO3- levels and an increase the concentration of essential amino acids, with all of them having beneficial properties for the human diet. © 2016 Society of Chemical Industry.

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues.

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants.

We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants.

Purification and biochemical characterisation of a glucose-6-phosphate dehydrogenase from the psychrophilic green alga Koliella antarctica.

Psychrophilic organisms have evolved a number of modifications of cellular structures to survive in the cold environment; among them it is worth noting an increased efficiency of enzymes at lower temperatures. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was purified and characterised from the psychrophilic green alga Koliella antarctica (Trebouxiophyceae, Chlorophyta) from the Ross Sea (Antarctica). It was possible to isolate a single G6PDH using biochemical strategies; its maximum activity was measured at 35 °C, and the enzyme showed an E (a) of 39.6 kJ mol(-1). This protein reacted with antibodies raised against higher plants plastidic isoforms. KaG6PDH showed peculiar kinetic properties, with a K (iNADPH) value lower than [Formula: see text]. Notably, catalytic activity was inactivated in vitro by DTT and chloroplastic thioredoxin f. These biochemical properties of G6PDH are discussed with respect to higher plant G6PDHs and the adaptation of K. antarctica to polar low-temperature environment.

Evaluation of the alkalinity stress tolerance of three Brassica rapa CAX1 TILLING mutants.

Alkalinity is an important environmental factor that affects crop production and will be exacerbated in the current climate change scenario. Thus, the presence of carbonates and high pH in soils negatively impacts nutrient assimilation and photosynthesis and causes oxidative stress. A potential strategy to improve tolerance to alkalinity could be the modification of cation exchanger (CAX) activity, given that these transporters are involved in calcium (Ca2+) signaling under stresses. In this study, we used three Brassica rapa mutants (BraA.cax1a-4, BraA.cax1a-7, and BraA.cax1a-12) from the parental line 'R-o-18' that were generated by Targeting Induced Local Lesions in Genomes (TILLING) and grown under control and alkaline conditions. The objective was to assess the tolerance of these mutants to alkalinity stress. Biomass, nutrient accumulation, oxidative stress, and photosynthesis parameters were analyzed. The results showed that BraA.cax1a-7 mutation was negative for alkalinity tolerance because it reduced plant biomass, increased oxidative stress, partially inhibited antioxidant response, and lowered photosynthesis performance. Conversely, the BraA.cax1a-12 mutation increased plant biomass and Ca2+ accumulation, reduced oxidative stress, and improved antioxidant response and photosynthesis performance. Hence, this study identifies BraA.cax1a-12 as a useful CAX1 mutation to enhance the tolerance of plants grown under alkaline conditions.

Plastic in the Environment: A Modern Type of Abiotic Stress for Plant Physiology.

In recent years, plastic pollution has become a growing environmental concern: more than 350 million tons of plastic material are produced annually. Although many efforts have been made to recycle waste, a significant proportion of these plastics contaminate and accumulate in the environment. A central point in plastic pollution is demonstrated by the evidence that plastic objects gradually and continuously split up into smaller pieces, thus producing subtle and invisible pollution caused by microplastics (MP) and nanoplastics (NP). The small dimensions of these particles allow for the diffusion of these contaminants in farmlands, forest, freshwater, and oceans worldwide, posing serious menaces to human, animal, and plant health. The uptake of MPs and NPs into plant cells seriously affects plant growth, development, and photosynthesis, finally limiting crop yields and endangering natural environmental biodiversity. Furthermore, nano- and microplastics-once adsorbed by plants-can easily enter the food chain, being highly toxic to animals and humans. This review addresses the impacts of MP and NP particles on plants in the terrestrial environment. In particular, we provide an overview here of the detrimental effects of photosynthetic injuries, oxidative stress, ROS production, and protein damage triggered by MN and NP in higher plants and, more specifically, in crops. The possible damage at the physiological and environmental levels is discussed.

Transcriptomic landscape of tomato traditional long shelf-life landraces under low water regimes.

'Corbarino' (COR) and 'Lucariello' (LUC) belong to the family of Mediterranean long shelf-life tomato landraces, producing high quality fruits under low water input cultivation regime in their traditional cultivation area. Understanding the morpho-physiological and molecular details of the peculiar drought stress tolerance of these two genotypes may be key to their valorization as breeding material. RNA sequencing of leaf samples of COR and LUC subjected to drought stress by water withholding in a semi-controlled greenhouse identified 3089 and 2135 differentially expressed genes respectively. These included COR- and LUC-specific annotated genes, as well as genes containing single nucleotide polymorphisms as compared to reference genome. Enriched Gene Ontology categories showed that categories such as response to water, oxidoreductase activity, nucleotide salvation and lipid biosynthesis-related processes were enriched among up-regulated DEGs. By contrast, growth and photosynthesis related genes were down-regulated after drought stress, consistent with leaf gas exchange and biomass accumulation measurements. Genes encoding cell wall degrading enzymes of the pectinase family were also down-regulated in drought stress conditions and upregulated in rewatering, indicating that cell wall composition/hardness is important for drought stress responses. Globally our results contribute to understanding the transcriptomic and physiological responses of representative tomato genotypes from Southern Italy, highlighting a promising set of genes to be investigated to improve tomato tolerance to drought.

Improvement of the physiological response of barley plants to both Zinc deficiency and toxicity by the application of calcium silicate.

An adequate availability of Zinc (Zn) is crucial for plant growth and development given the essentiality of this element. Thus, both Zn deficiency and Zn toxicity can limit crop yields. In plants, the responses to Zn imbalances involve important physiological aspects such as reactive oxygen species (ROS) accumulation, phytohormone balance, tricarboxylic acid cycle (TCA) metabolism, and organic acids (OAs) accumulation. However, a way to improve tolerance to stresses such as those produced by nutritional imbalances is the application of beneficial elements such as silicon (Si). In this study, we grew barley plants in hydroponics under Zn deficiency and toxicity conditions, applying Si in the form of CaSiO3 in order to assess its effectiveness against Zn imbalances. Parameters related to plant growth, oxidative stress, TCA enzyme activities, phytohormones and OAs accumulation were analyzed. Both Zn deficiency and toxicity reduced leaf biomass, increased ROS accumulation, and affected phytohormone and OAs concentrations and TCA enzyme activities. CaSiO3 treatment was effective in counteracting these effects enhancing Zn accumulation under Zn deficient conditions and limiting its accumulation under toxic conditions. In addition, this treatment decreased ROS levels, and improved ascorbate/glutathione and phytohormonal responses, citrate synthase activity, and malate/oxalate ratio. Therefore, this study enhanced the notion of the efficacy of CaSiO3 in improving tolerance to Zn imbalances.

Photosynthetic, Molecular and Ultrastructural Characterization of Toxic Effects of Zinc in Caulerpa racemosa Indicate Promising Bioremediation Potentiality.

Caulerpaceae are unconventional green algae composed of multinucleated, single siphonous cells. The species of Caulerpa are acquiring major scientific interest for both their invasion in the Mediterranean ecological niche and for the production of valuable natural metabolites. Furthermore, the abilities of Caulerpa spp. in the biorecovery of polluted waters were recently investigated. Among heavy metal contaminants in marine systems, zinc (Zn) is considered a critical pollutant, progressively accumulating from plastic leachates. In this study, the responses of Caulerpa racemosa to different levels (5-10 mg L-1) of Zn were studied for 14 days under laboratory-controlled conditions. Effects of Zn were monitored by measuring the growth rate, photosynthetic efficiency and gene expression. Moreover, the ability of Caulerpa to remove Zn from seawater was monitored. Zn induced detrimental effects by decreasing the relative growth rate (RGR) and maximal PSII photochemical efficiency (Fv/Fm). Moreover, C. racemosa, grown in contaminated seawater, reduced the levels of Zn to a final concentration of 1.026 and 1.932 mg L-1 after 14 days, thus demonstrating efficient uptake. Therefore, our results characterized the effects of zinc on C. racemosa and the possible role of this alga as being effective in the bioremediation of marine seawater.

Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus.

Plants respond to changes of nutrient availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis (Arabidopsis thaliana) root development represents a paradigm of these responses, and nitrate transporters are involved both in local and systemic control. Ammonium (NH(4)(+)) represents an important nitrogen (N) source for plants, although toxicity symptoms are often associated with high NH(4)(+) concentration when this is present as the only N source. The reason for these effects is still controversial, and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect, suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH(4)(+) conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short-root phenotype was phenocopied in transgenic lines by LjAMT1;3 overexpression independently of ammonium supply, and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response.

Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase.

Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants.

Advanced Applications for Protein and Compounds from Microalgae.

Algal species still show unrevealed and unexplored potentiality for the identification of new compounds. Photosynthetic organisms represent a valuable resource to exploit and sustain the urgent need of sustainable and green technologies. Particularly, unconventional organisms from extreme environments could hide properties to be employed in a wide range of biotechnology applications, due to their peculiar alleles, proteins, and molecules. In this review we report a detailed dissection about the latest and advanced applications of protein derived from algae. Furthermore, the innovative use of modified algae as bio-reactors to generate proteins or bioactive compounds was discussed. The latest progress about pharmaceutical applications, including the possibility to obtain drugs to counteract virus (as SARS-CoV-2) were also examined. The last paragraph will survey recent cases of the utilization of extremophiles as bio-factories for specific protein and molecule production.