Histidinol-phosphate aminotransferase is the sixth protein (hence HISN6) in the histidine biosynthetic pathway in plants. HISN6 is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of imidazole acetol phosphate into L-histidinol phosphate (HOLP). Here, we show that plant HISN6 enzymes are closely related to the orthologs from Chloroflexota. The studied example, HISN6 from Medicago truncatula (MtHISN6), exhibits a surprisingly high affinity for HOLP, which is much higher than reported for bacterial homologs. Moreover, unlike the latter, MtHISN6 does not transaminate phenylalanine. High-resolution crystal structures of MtHISN6 in the open and closed states, as well as the complex with HOLP and the apo structure without PLP, bring new insights into the enzyme dynamics, pointing at a particular role of a string-like fragment that oscillates near the active site and participates in the HOLP binding. When MtHISN6 is compared to bacterial orthologs with known structures, significant differences arise in or near the string region. The high affinity of MtHISN6 appears linked to the particularly tight active site cavity. Finally, a virtual screening against a library of over 1.3 mln compounds revealed three sites in the MtHISN6 structure with the potential to bind small molecules. Such compounds could be developed into herbicides inhibiting plant HISN6 enzymes absent in animals, which makes them a potential target for weed control agents.

Carbon Farming is a new approach for land management that allows the sequestration of carbon in the soil and plants, mitigating the emission of greenhouse gases. It includes a set of eco-friendly strategies. Among them, increasing of carbon content through the application of biochar seems to be particularly promising for the Mediterranean area, where erosion and loss of organic matter is a relevant problem for agricultural production. Although soil amendment with biochar has received significant and increasing attention during the last decade, few studies have been focused on the Mediterranean basin. In this review, state of the art use of biochar as carbon farming practice in the Mediterranean region from the legislative, policy and scientific point of view has been discussed. Throughout the review, a description of the main results obtained from 36 studies carried out at 23 different field locations, with regard to carbon dynamics, soil water holding capacity (WHC) and crop yield has been reported. As a concluding remark, it is observed that biochar has good potential to store carbon in Medi-terranean soils; it has a good proportion of recalcitrant carbon and can effectively improve soil water content under limited water conditions such as during drought and summer. However, long-term field studies on various Mediterranean soils should be performed in order to reach valid conclusions on the effects of biochar on the plant-soil system in the Mediterranean basin. In this context, it is essential to report a set of basic parameters to link the main characteristics of biochar, and its effects on soil and plant productivity.

Melilotus officinalis is a Leguminosae with relevant applications in medicine and soil recovery. This study reports the application of Melilotus officinalis plants in soil recovery and as a source of bioactive compounds. Plants were cultivated in semiarid soil under four different fertilizer treatments, urban waste compost at 10 t/ha and 20 t/ha, inorganic fertilizer and a control (no fertilizer). Agronomic properties of soil (pH, EC, soil respiration, C content, macro- and microelements) were analyzed before and after treatment. Also, germination, biomass, element contents, and physiological response were evaluated. Metabolite composition of plants was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Results showed a significant enhancement of the soil microbial activity in planted soils amended with compost, though there were no other clear effects on the soil physicochemical and chemical characteristics during the short experimental period. An improvement in M. officinalis germination and growth was observed in soils with compost amendment. Metabolite composition of plants was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Principal Component and Agglomerative Hierarchical Clustering models suggest that there is a clear separation of the metabolome of four groups of plants grown under different soil treatments. The five most important discriminative metabolites (annotated) were oleamide, palmitic acid, stearic acid, 3-hydroxy-cis-5-octenoylcarnitine, and 6-hydroxynon-7- enoylcarnitine. This study provides information on how the metabolome of Melilotus might be altered by fertilizer application in poor soil regions. These metabolome changes might have repercussions for the application of this plant in medicine and pharmacology. The results support the profitability of Melilotus officinalis cultivation for bioactive compounds production in association with soil recovery practices.

Although the use of composts derived from anaerobic digestates as soil amendments is likely to increase in the future, there is little information concerning the fate of their C and N compounds after their incorporation into soil. This work assesses C and N concentrations and the associated changes in ?N and ?C during the composting processes of cattle and pig slurry anaerobic digestates. In addition, the compost effect on C and N fractions and plant uptake were studied during a six-month pot experiment with rosemary plants. The results did not show ?C and ?N isotopic discrimination during composting, indicating a previous stabilization of cattle manure and pig slurry during the anaerobic digestion. This fact was also confirmed by the low C losses during the composting processes (1.2-fold and 1.05-fold for the composting piles with cattle and pig slurry anaerobic digestates, respectively). After soil addition, the composts augmented N values (from 0.41 g kg to around 0.56 g kg in low dose and 0.68 g kg in high dose compost amended soils) and ?N soil values (increases in the range of 50%-156%), but showed only slight differences in C and ?C values compared to unfertilised control and inorganic fertilized soils. Moreover, the rosemary leaves of the plants grown on the compost amended soils presented higher N and ?N abundance than control and inorganic fertilized plants. We conclude that ?N abundance of anaerobic digestate composts is useful to discern its N uptake and could thus be a useful tool to detect whether organic or mineral fertiliser types were used for agricultural production.

Serine hydroxymethyltransferase (SHM) is one of the hallmarks of one-carbon metabolism. In plants, isoforms of SHM participate in photorespiration and/or transfer the one-carbon unit from L-serine to tetrahydrofolate (THF), hence producing 5,10-CH-THF that is needed, e.g., for biosynthesis of methionine, thymidylate, and purines. These links highlight the importance of SHM activity in DNA biogenesis, its epigenetic methylations, and in stress responses. Plant genomes encode several SHM isoforms that localize to cytosol, mitochondria, plastids, and nucleus. In this work, we present a thorough functional and structural characterization of all seven SHM isoforms from Arabidopsis thaliana (AtSHM1-7). In particular, we analyzed tissue-specific expression profiles of the AtSHM genes. We also compared catalytic properties of the active AtSHM1-4 in terms of catalytic efficiency in both directions and inhibition by the THF substrate. Despite numerous attempts to rescue the SHM activity of AtSHM5-7, we failed, which points towards different physiological functions of these isoforms. Comparative analysis of experimental and predicted three-dimensional structures of AtSHM1-7 proteins indicated differences in regions that surround the entrance to the active site cavity.

Vitamins B (thiamine) and B (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5?-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5?-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg but activating the Zn-dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B- and B-dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in K of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased K of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness. (Figure presented.).

The disturbed metabolism of vitamins B1 or B6, which are essential for neurotransmitters homeostasis, may cause seizures. Our study aims at revealing therapeutic potential of vitamins B1 and B6 by estimating the short- and long-term effects of their combined administration with the seizure inductor pentylenetetrazole (PTZ). The PTZ dose dependence of a seizure and its parameters according to modified Racine's scale, along with delayed physiological and biochemical consequences the next day after the seizure are assessed regarding sexual dimorphism in epilepsy. PTZ sensitivity is stronger in the female than the male rats. The next day after a seizure, sex differences in behavior and brain biochemistry arise. The induced sex differences in anxiety and locomotor activity correspond to the disappearance of sex differences in the brain aspartate and alanine, with appearance of those in glutamate and glutamine. PTZ decreases the brain malate dehydrogenase activity and urea in the males and the phenylalanine in the females. The administration of vitamins B1 and B6 24 h before PTZ delays a seizure in female rats only. This desensitization is not observed at short intervals (0.5-2 h) between the administration of the vitamins and PTZ. With the increasing interval, the pyridoxal kinase (PLK) activity in the female brain decreases, suggesting that the PLK downregulation by vitamins contributes to the desensitization. The delayed effects of vitamins and/or PTZ are mostly sex-specific and interacting. Our findings on the sex differences in sensitivity to epileptogenic factors, action of vitamins B1/B6 and associated biochemical events have medical implications.

This paper reports the application of organic amendments, such as biochar (BC) or compost (CMP) in a Plant-assisted bioremediation (PABR) strategy of a soil contaminated by polychlorinated biphenyls (PCBs) and heavy metals (HM). BC and CMP were applied for improving PABR in specific plots where the highest amounts of PCBs were present. Before the organic treatments and six months after it, soil samples and plant tissue (leaves, shoots and roots) were collected from each investigated plot and chemical and microbiological analyses performed. Moreover, micro-X ray fluorescence (µXRF) analysis was performed on soil and biomass (leaves, roots). Preliminary results evidenced different effects of the CMP or BC on promoting PCB and HM removal, depending on the initial pollutant concentrations

Defects of vitamin B metabolism are responsible for severe neurological disorders, such as pyridoxamine 5?-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5?-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.

Rosmarinus officinalis is an evergreen aromatic plant with important commercial interest as it contains numerous essential oils (composed of terpenoid compounds) and phenolic constituents (natural antioxidant compounds). This work aims at evaluating the concomitant effects of different inorganic and organic fertilization treatments and the subsequent increases in soil nutrient availability on terpenoids and other carbon-based secondary metabolites, e.g., flavonoids and phenolic compounds, in Rosmarinus officinalis leaves. The results showed that, as expected, the structural carbohydrate content (lignocellulosic compounds) in stems was higher in fertilized plants than in controls. Additionally, positive correlations were observed of the absolute amounts of total terpenoids and some single terpenoid compounds with N or P contents in leaves. On the contrary, the phenolic and flavonoid concentrations in all the rosemary plant parts were lower with the fertilization treatments. Indeed, negative correlations between the phenolic compounds (and flavonoids) and N in rosemary leaves were also found. Overall, the results suggest that the terpenoid production's response to fertilization was due to N, which is essential for protein synthesis and terpene synthase activity, and to P, which is necessary for the synthesis of both terpenoid precursors and ATP and NADPH, also needed for terpenoid synthesis. On the other hand, the basis for the fertilization's effects on the production of phenolic compounds is the direct nitrogen trade-off between growth and the shikimic acid pathway by which phenolics compounds are synthesized.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5'-phosphate-dependent enzyme that plays a pivotal role in cellular one-carbon metabolism. In plants and cyanobacteria, this enzyme is also involved in photorespiration and confers salt tolerance, as in the case of SHMT from the halophilic cyanobacterium Aphanothece halophytica (AhSHMT). We have characterized the catalytic properties of AhSHMT in different salt and pH conditions. Although the kinetic properties of AhSHMT correlate with those of the mesophilic orthologue from Escherichia coli, AhSHMT appears more catalytically efficient, especially in presence of salt. Our studies also reveal substrate inhibition, previously unobserved in AhSHMT. Furthermore, addition of the osmoprotectant glycine betaine under salt conditions has a distinct positive effect on AhSHMT activity. The crystal structures of AhSHMT in three forms, as internal aldimine, as external aldimine with the l-serine substrate, and as a covalent complex with malonate, give structural insights on the possible role of specific amino acid residues implicated in the halophilic features of AhSHMT. Importantly, we observed that overexpression of the gene encoding SHMT, independently from its origin, increases the capability of E. coli to grow in high salt conditions, suggesting that the catalytic activity of this enzyme in itself plays a fundamental role in salt tolerance.

In Escherichia coli, the synthesis of pyridoxal 5?-phosphate (PLP), the catalytically active form of vitamin B-6, takes place through the so-called deoxyxylulose 5-phosphate?dependent pathway, whose last step is pyridoxine 5?-phosphate (PNP) oxidation to PLP, catalyzed by the FMN-dependent enzyme PNP oxidase (PNPOx). This enzyme plays a pivotal role in controlling intracellular homeostasis and bioavailability of PLP. PNPOx has been proposed to undergo product inhibition resulting from PLP binding at the active site. PLP has also been reported to bind tightly at a secondary site, apparently without causing PNPOx inhibition. The possible location of this secondary site has been indicated by crystallographic studies as two symmetric surface pockets present on the PNPOx homodimer, but this site has never been verified by other experimental means. Here, we demonstrate, through kinetic measurements, that PLP inhibition is actually of a mixed-type nature and results from binding of this vitamer at an allosteric site. This interpretation was confirmed by the characterization of a mutated PNPOx form, in which substrate binding at the active site is heavily hampered but PLP binding is preserved. Structural and functional connections between the active site and the allosteric site were indicated by equilibrium binding experiments, which revealed different PLP-binding stoichiometries with WT and mutant PNPOx forms. These observations open up new horizons on the mechanisms that regulate E. coli PNPOx, which may have commonalities with the mechanisms regulating human PNPOx, whose crucial role in vitamin B-6 metabolism and epilepsy is well-known.

Serine hydroxymethyltransferases (SHMTs) reversibly transform serine into glycine in a reaction accompanied with conversion of tetrahydrofolate (THF) into 5,10-methylene-THF (5,10-meTHF). In vivo, 5,10-meTHF is the main carrier of one-carbon (1C) units, which are utilized for nucleotide biosynthesis and other processes crucial for every living cell, but hyperactivated in overproliferating cells (e.g. cancer tissues). SHMTs are emerging as a promising target for development of new drugs because it appears possible to inhibit growth of cancer cells by cutting off the supply of 5,10-meTHF. Methotrexate (MTX) and pemetrexed (PTX) are two examples of antifolates that have cured many patients over the years but target different enzymes from the folate cycle (mainly dihydrofolate reductase and thymidylate synthase, respectively). Here we show crystal structures of MTX and PTX bound to plant SHMT isozymes from cytosol and mitochondria-human isozymes exist in the same subcellular compartments. We verify inhibition of the studied isozymes by a thorough kinetic analysis. We propose to further exploit antifolate scaffold in development of SHMT inhibitors because it seems likely that especially polyglutamylated PTX inhibits SHMTs in vivo. Structure-based optimization is expected to yield novel antifolates that could potentially be used as chemotherapeutics.

Polychlorinated byphenyls (PCBs) are a class of Persistent Organic Pollutants extremely hard to remove from soil. The use of plants to promote the degradation of PCBs, thanks to synergic interactions between roots and the natural soil microorganisms in the rhizosphere, has been proved to constitute an eective and environmentally friendly remediation technique. Preliminary microcosm experiments were conducted in a greenhouse for 12 months to evaluate the capacity of the Monviso hybrid poplar clone, a model plant for phytoremediation, to grow in a low quality and PCB-contaminated soil in order to assess if this clone could be subsequently used in a field experiment. For this purpose, three dierent soil conditions (Microbiologically Active, Pre-sterilized and Hypoxic soils) were set up in order to assess the capacity of this clone to grow in the polluted soil in these dierent conditions and support the soil microbial community activity. The growth and physiology (chlorophyll content, chlorophyll fluorescence, ascorbate, phenolic compounds and flavonoid contents) of the poplar were determined. Moreover, chemical analyses were performed to assess the concentrations of PCB indicators in soil and plant roots. Finally, the microbial community was evaluated in terms of total abundance and activity under the dierent experimental conditions. Results showed that the poplar clone was able to grow eciently in the contaminated soil and to promote microbial transformations of PCBs. Plants grown in the hypoxic condition promoted the formation of a higher number of higher-chlorinated PCBs and accumulated lower PCBs in their roots. However, plants in this condition showed a higher stress level than the other microcosms, producing higher amounts of phenolic, flavonoid and ascorbate contents, as a defence mechanism. Keywords: natural-based remediation strategies; Monviso clone; plant physiology; antioxidant

The study reports on the effect of anaerobic digestate derived composts on the metabolite composition and thermal behaviour of rosemary (Rosmarinus officinalis L.). Plants were cultivated in semiarid soil under four different fertiliser treatments (composts of anaerobic digested cattle (C) or pig slurry (P) at 30t/ha and 60 t/ha, and two control treatments (inorganic fertiliser and no fertiliser application). Samples of leaves and stems were analysed to investigate the effect of treatment on chemical composition and thermochemical properties. Three orthogonal analytical approaches were used, namely: Fourier transform mid infrared spectroscopy (FTIR), gas chromatography/mass spectrometry (GC/MS) and thermochemical gravimetric analysis (TGA). FTIR and GC/MS showed fertiliser treatment resulted in tissue specific changes in sample metabolite composition. Fertiliser treatment was detected to change the thermogravimetric properties of the leaf samples and from inorganic and composted pig slurry digestate treatments had greater ash content and lower proportions of fixed carbon compared with samples from the unfertilised control treatment. This study provides information on how the composition of rosemary might be altered by fertiliser application in regions of poor soil, and has implications for biomass quality when rosemary is grown on semi-wild sites for the purpose of soil improvement

Effects of supplemented UV radiation and diminished water supply on the leaf concentrations of phenols and antioxidants of two Mediterranean resprouter species, Arbutus unedo and Quercus suber, were assessed before and after entire aerial biomass removal. Potted seedlings of both species were grown outdoors for 8 months with enhanced UV-A + UV-B, enhanced UV-A or ambient UV, in combination with two watering conditions (field capacity or watering reduction). After this period, all aerial biomass was removed and new shoots (resprouts) developed for a further 8 months under the two treatments. In general, the investment in leaf phenols was substantially greater in A. unedo than in Q. suber, while Q. suber allocated more resources to non-phenolic antioxidants (ascorbate and glutathione). In response to enhanced UV-B radiation, Q. suber leaves rose their UV-screening capacity mainly via accumulation of kaempferols, accompanied by an increased concentration of rutins, being these effects exacerbated under low-watering conditions. Conversely, A. unedo leaves responded to UV-B radiation reinforcing the antioxidant machinery by increasing the overall amount of flavonols (especially quercetins) in seedlings, and of ascorbate and glutathione, along with catalase activity, in resprouts. Nevertheless, UV effects on the amount/activity of non-phenolic antioxidants of A. unedo resprouts were modulated by water supply. Indeed, the highest concentration of glutathione was found under the combination of enhanced UV-B radiation and reduced watering, suggesting an enlargement of the antioxidant response in A. unedo resprouts. Different biochemical responses to enhanced UV and drier conditions in seedlings and resprouts of these two species might modulate their competitive interactions in the near future.

Climate change projections forecast an average warming in mean temperature and an associated change in the distribution and intensity of rainfalls. At the manipulation experiment in Kiskunság, Hungary, we aimed to study the effect of the soil drought and night-time warming conditions projected for the coming decades on leaf gas exchange, including photosynthetic rates and isoprene emission rates and on isoprene synthase expression levels of Populus alba sprouts (root suckers). During the years 2010 and 2011, warming treatment increased the air temperature 0.35 °C on average whereas drought treatment reduced the soil moisture by 13% on average in relation to control plots. The results highlighted important seasonal differences. Photosynthesis was limited by stomatal closure in summer and also isoprene emission and isoprene synthase expression levels were lower in summer than in autumn for all treatments. As a consequence, a negative relationship was found between temperature, ranging from 25 °C to 35 °C, and isoprene emission throughout all the study period and treatments. Results also showed significant treatments effects on isoprene parameters but only in July 2011. Isoprene emission decreased with drought, and isoprene synthase expression levels decreased with night-time warming. A positive relationship was found between isoprene emission and leaf internal carbon in drought treated poplar plants, which contrasted with our findings for control and warming treated plants. These results indicated either a change in the regulation of pyruvate distribution between isoprene synthesis and respiration under drought or a utilization of alternative carbon sources for isoprene emission.