C3H Expression Is Crucial for Methyl Jasmonate Induction of Chicoric Acid Production by Echinacea purpurea (L.) Moench Cell Suspension Cultures.

Echinacea purpurea (L.) Moench is one of the most economically important medicinal plants, cultivated worldwide for its high medicinal value and with several industrial applications in both pharmaceutical and food industries. Thanks to its various phytochemical contents, including caffeic acid derivatives (CADs), E. purpurea extracts have antioxidant, anti-inflammatory, and immuno-stimulating properties. Among CADs, chicoric acid is one of the most important compounds which have shown important pharmacological properties. The present research was aimed at optimizing the production of chicoric acid in E. purpurea cell culture. Methyl jasmonate (MeJa) at different concentrations and for different duration of treatments was utilized as elicitor, and the content of total polyphenols and chicoric acid was measured. Several genes involved in the chicoric acid biosynthetic pathway were selected, and their expression evaluated at different time points of cell culture growth. This was performed with the aim of identifying the most suitable putative molecular markers to be used as a proxy for the early prediction of chicoric acid contents, without the need of expensive quantification methods. A correlation between the production of chicoric acid in response to MeJa and an increased response to oxidative stress was also proposed.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

Nitrate Regulates Maize Root Transcriptome through Nitric Oxide Dependent and Independent Mechanisms.

Maize root responds to nitrate by modulating its development through the coordinated action of many interacting players. Nitric oxide is produced in primary root early after the nitrate provision, thus inducing root elongation. In this study, RNA sequencing was applied to discover the main molecular signatures distinguishing the response of maize root to nitrate according to their dependency on, or independency of, nitric oxide, thus discriminating the signaling pathways regulated by nitrate through nitric oxide from those regulated by nitrate itself of by further downstream factors. A set of subsequent detailed functional annotation tools (Gene Ontology enrichment, MapMan, KEGG reconstruction pathway, transcription factors detection) were used to gain further information and the lateral root density was measured both in the presence of nitrate and in the presence of nitrate plus cPTIO, a specific NO scavenger, and compared to that observed for N-depleted roots. Our results led us to identify six clusters of transcripts according to their responsiveness to nitric oxide and to their regulation by nitrate provision. In general, shared and specific features for the six clusters were identified, allowing us to determine the overall root response to nitrate according to its dependency on nitric oxide.

Nitrate and Ammonium Affect the Overall Maize Response to Nitrogen Availability by Triggering Specific and Common Transcriptional Signatures in Roots.

Nitrogen (N) is an essential macronutrient for crops. Plants have developed several responses to N fluctuations, thus optimizing the root architecture in response to N availability. Nitrate and ammonium are the main inorganic N forms taken up by plants, and act as both nutrients and signals, affecting gene expression and plant development. In this study, RNA-sequencing was applied to gain comprehensive information on the pathways underlying the response of maize root, pre-treated in an N-deprived solution, to the provision of nitrate or ammonium. The analysis of the transcriptome shows that nitrate and ammonium regulate overlapping and distinct pathways, thus leading to different responses. Ammonium activates the response to stress, while nitrate acts as a negative regulator of transmembrane transport. Both the N-source repress genes related to the cytoskeleton and reactive oxygen species detoxification. Moreover, the presence of ammonium induces the accumulation of anthocyanins, while also reducing biomass and chlorophyll and flavonoids accumulation. Furthermore, the later physiological effects of these nutrients were evaluated through the assessment of shoot and root growth, leaf pigment content and the amino acid concentrations in root and shoot, confirming the existence of common and distinct features in response to the two nitrogen forms.

Triterpene Acid and Phenolics from Ancient Apples of Friuli Venezia Giulia as Nutraceutical Ingredients: LC-MS Study and In Vitro Activities.

Triterpene acid and phenolic constituents from nine ancient varieties of apple (Malus domestica) fruits cultivated in Fanna, Friuli Venezia Giulia region, northeast Italy, were analyzed and compared with four commercial apples ('Golden Delicious', 'Red Delicious', 'Granny Smith' and 'Royal Gala'). Total phenolic and flavonoid contents were measured by spectrophotometric assays. The quali-quantitative fingerprint of secondary metabolites including triterpene acid was obtained by LC-DAD-(ESI)-MS and LC-(APCI)-MS, respectively. Based on the two LC-MS datasets, multivariate analysis was used to compare the composition of ancient fruit varieties with those of four commercial apples. Significant differences related mainly to the pattern of triterpene acids were found. Pomolic, euscaphyc, maslinic and ursolic acids are the most abundant triterpene in ancient varieties pulps and peels, while ursolic and oleanolic acids were prevalent in the commercial fruits. Also, the content of the phenolic compounds phloretin-2-O-xyloglucoside and quercetin-3-O-arabinoside was greater in ancient apple varieties. The antioxidant (radical scavenging, reducing power, metal chelating and phosphomolybdenum assays) and enzyme inhibitory effects (against cholinesterase, tyrosinase, amylase and glucosidase) of the samples were investigated in vitro. Antioxidant assays showed that the peels were more active than pulps. However, all the samples exhibited similar enzyme inhibitory effects. Ancient Friuli Venezia Giulia apple cultivars can be a source of chlorogenic acid and various triterpene acids, which are known for their potential anti-inflammatory activity and beneficial effects on lipid and glucose metabolism. Our results make these ancient varieties suitable for the development of new nutraceutical ingredients.

Preliminary evaluation of quince (Cydonia oblonga Mill.) fruit as extraction source of antioxidant phytoconstituents for nutraceutical and functional food applications.

BACKGROUND: Quince (Cydonia oblonga) fruits can be considered as starting material for the extraction of health-promoting phytochemicals, to be exploited in food and nutraceuticals. In the present work, liquid chromatography coupled with diode array detection and tandem mass spectrometry analysis allowed the study of the phytochemical composition of quince fruits and to compare it with those of six commercial apple varieties. RESULTS: The distribution and quantification of secondary metabolites in peel and pulp were studied and compared with six commercial apple varieties. Furthermore the in vitro antioxidant activity was determined by 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay. Quince fruit presented significant amounts of shikimic and quinic acid derivatives, as well as flavonoids and procyanidins. Compared with apple, quince fruit composition was characterized by the presence of 4-caffeoylshikimic acid, 4-caffeoyl quinic acid, quercetin-3,7-diglucoside, kaempferol-3-O-rhamnoside and kaempferol-7-O-glucoside, and the dihydrochalcones were not detectable. The peel showed the highest contents of phenolics, whereas 3-O-caffeoylquinic acid was the most abundant compound in the quince pulp. The Pearson correlation index was calculated considering the quantitative amount of the phenolic constituents and the radical scavenging activity toward DPPH· both for peel and pulp extracts. CONCLUSIONS: This study highlighted the presence of significant amounts of valuable secondary metabolites in quince fruit, in particular the procyanidins and caffeoyl esters with shikimic and quinic acid. Notably, owing to the higher content in phenolic compounds and the stronger antioxidant capacity compared with the other fruits considered, the use of C. oblonga as a source of antioxidant can be valuable in nutraceuticals, revealing new possible applications of quince fruit. © 2018 Society of Chemical Industry.

Supercritical CO2 Extraction of Eruca sativa Using Cosolvents: Phytochemical Composition by LC-MS Analysis.

BACKGROUND: Eruca sativa Mill. is a good source of glucosinolates (GLS), phenolic compounds and unsaturated fatty acids, being a valuable material for the production of functional-foods or nutraceutical ingredients. Extraction by supercritical CO2 (SCO2) can be used and the limitations due to the apolar nature of CO2 can be overcome using co-solvents. In this paper different cosolvents and conditions were used for SCO2 extraction and the composition of the obtained extracts was studied by LC-MS. RESULTS: Water resulted the ideal co-solvent, allowing the extraction of glucosinolates in comparable amounts to the classical procedure with boiling water, as it can be carried out at mild temperatures (45 °C vs. >100 °C). Increasing the pressure improved the GLS extraction. On the other hand polyphenol extraction under the studied conditions was not influenced by pressure and temperature variations. The in vitro antioxidant effect of the obtained extracts was also measured, showing significant activity in the DPPH and FC tests. CONCLUSIONS: The GLS, flavonoids and lipids composition of the obtained extracts was studied, showing the presence of numerous antioxidant constituents useful for nutraceutical applications. The extraction method using SCO2 and water as co-solvent presents advantages in terms of safety because these solvents are generally recognised as safe. Water as cosolvent at 8% resulted useful for the extraction of both glucosinolates and phenolics in good amount and is environmentally acceptable as well as safe for food production.

System analysis of metabolism and the transcriptome in Arabidopsis thaliana roots reveals differential co-regulation upon iron, sulfur and potassium deficiency.

Deprivation of mineral nutrients causes significant retardation of plant growth. This retardation is associated with nutrient-specific and general stress-induced transcriptional responses. In this study, we adjusted the external supply of iron, potassium and sulfur to cause the same retardation of shoot growth. Nevertheless, limitation by individual nutrients resulted in specific morphological adaptations and distinct shifts within the root metabolite fingerprint. The metabolic shifts affected key metabolites of primary metabolism and the stress-related phytohormones, jasmonic, salicylic and abscisic acid. These phytohormone signatures contributed to specific nutrient deficiency-induced transcriptional regulation. Limitation by the micronutrient iron caused the strongest regulation and affected 18% of the root transcriptome. Only 130 genes were regulated by all nutrients. Specific co-regulation between the iron and sulfur metabolic routes upon iron or sulfur deficiency was observed. Interestingly, iron deficiency caused regulation of a different set of genes of the sulfur assimilation pathway compared with sulfur deficiency itself, which demonstrates the presence of specific signal-transduction systems for the cross-regulation of the pathways. Combined iron and sulfur starvation experiments demonstrated that a requirement for a specific nutrient can overrule this cross-regulation. The comparative metabolomics and transcriptomics approach used dissected general stress from nutrient-specific regulation in roots of Arabidopsis.

Use of oleaginous plants in phytotreatment of grey water and yellow water from source separation of sewage.

Efficient and economic reuse of waste is one of the pillars of modern environmental engineering. In the field of domestic sewage management, source separation of yellow (urine), brown (faecal matter) and grey waters aims to recover the organic substances concentrated in brown water, the nutrients (nitrogen and phosphorous) in the urine and to ensure an easier treatment and recycling of grey waters. With the objective of emphasizing the potential of recovery of resources from sewage management, a lab-scale research study was carried out at the University of Padova in order to evaluate the performances of oleaginous plants (suitable for biodiesel production) in the phytotreatment of source separated yellow and grey waters. The plant species used were Brassica napus (rapeseed), Glycine max (soybean) and Helianthus annuus (sunflower). Phytotreatment tests were carried out using 20L pots. Different testing runs were performed at an increasing nitrogen concentration in the feedstock. The results proved that oleaginous species can conveniently be used for the phytotreatment of grey and yellow waters from source separation of domestic sewage, displaying high removal efficiencies of nutrients and organic substances (nitrogen>80%; phosphorous >90%; COD nearly 90%). No inhibition was registered in the growth of plants irrigated with different mixtures of yellow and grey waters, where the characteristics of the two streams were reciprocally and beneficially integrated.

Drought stress in maize causes differential acclimation responses of glutathione and sulfur metabolism in leaves and roots.

BACKGROUND: Drought is the most important environmental stress that limits crop yield in a global warming world. Despite the compelling evidence of an important role of oxidized and reduced sulfur-containing compounds during the response of plants to drought stress (e.g. sulfate for stomata closure or glutathione for scavenging of reactive oxygen species), the assimilatory sulfate reduction pathway is almost not investigated at the molecular or at the whole plant level during drought. RESULTS: In the present study, we elucidated the role of assimilatory sulfate reduction in roots and leaves of the staple crop maize after application of drought stress. The time-resolved dynamics of the adaption processes to the stress was analyzed in a physiological relevant situation -when prolonged drought caused significant oxidation stress but root growth should be maintained. The allocation of sulfate was significantly shifted to the roots upon drought and allowed for significant increase of thiols derived from sulfate assimilation in roots. This enabled roots to produce biomass, while leaf growth was stopped. Accumulation of harmful reactive oxygen species caused oxidation of the glutathione pool and decreased glutathione levels in leaves. Surprisingly, flux analysis using [35S]-sulfate demonstrated a significant down-regulation of sulfate assimilation and cysteine synthesis in leaves due to the substantial decrease of serine acetyltransferase activity. The insufficient cysteine supply caused depletion of glutathione pool in spite of significant transcriptional induction of glutathione synthesis limiting GSH1. Furthermore, drought impinges on transcription of membrane-localized sulfate transport systems in leaves and roots, which provides a potential molecular mechanism for the reallocation of sulfur upon prolonged water withdrawal. CONCLUSIONS: The study demonstrated a significant and organ-specific impact of drought upon sulfate assimilation. The sulfur metabolism related alterations at the transcriptional, metabolic and enzyme activity level are consistent with a promotion of root growth to search for water at the expense of leaf growth. The results provide evidence for the importance of antagonistic regulation of sulfur metabolism in leaves and roots to enable successful drought stress response at the whole plant level.

Metabolic and Molecular Rearrangements of Sauvignon Blanc (Vitis vinifera L.) Berries in Response to Foliar Applications of Specific Dry Yeast.

Dry yeast extracts (DYE) are applied to vineyards to improve aromatic and secondary metabolic compound content and wine quality; however, systematic information on the underpinning molecular mechanisms is lacking. This work aimed to unravel, through a systematic approach, the metabolic and molecular responses of Sauvignon Blanc berries to DYE treatments. To accomplish this, DYE spraying was performed in a commercial vineyard for two consecutive years. Berries were sampled at several time points after the treatment, and grapes were analyzed for sugars, acidity, free and bound aroma precursors, amino acids, and targeted and untargeted RNA-Seq transcriptional profiles. The results obtained indicated that the DYE treatment did not interfere with the technological ripening parameters of sugars and acidity. Some aroma precursors, including cys-3MH and GSH-3MH, responsible for the typical aromatic nuances of Sauvignon Blanc, were stimulated by the treatment during both vintages. The levels of amino acids and the global RNA-seq transcriptional profiles indicated that DYE spraying upregulated ROS homeostatic and thermotolerance genes, as well as ethylene and jasmonic acid biosynthetic genes, and activated abiotic and biotic stress responses. Overall, the data suggested that the DYE reduced berry oxidative stress through the regulation of specific subsets of metabolic and hormonal pathways.

Foliar Application of Silicon in Vitis vinifera: Targeted Metabolomics Analysis as a Tool to Investigate the Chemical Variations in Berries of Four Grapevine Cultivars.

Silicon (Si) is a beneficial element for the growth of various crops, but its effect on plant metabolism is still not completely elucidated. Even if Si is not classified as an essential element for plants, the literature has reported its beneficial effects in a variety of species. In this work, the influence of Si foliar application on berry composition was evaluated on four grapevine cultivars. The berries of Teroldego and Oseleta (red grapes) and Garganega and Chardonnay (white grapes) were analyzed after foliar application of silicon by comparing the treated and control groups. A targeted metabolomic approach was used that focused on secondary metabolites, amino acids, sugars, and tartaric acid. Measurements were performed using liquid chromatography coupled with a diode array detector and mass spectrometry (LC-DAD-MSn), a LC-evaporative light scattering detector (ELDS), and LC-MS/MS methods specific for the analysis of each class of constituents. After the data collection, multivariate models, PCA, PLS-DA, OPLS-DA, were elaborated to evaluate the effect of Si application in the treated vs. control samples. Results were different for each grape cultivar. A significant increase in anthocyanins was observed in the Oseleta cultivar, with 0.48 mg g-1 FW in the untreated samples vs. 1.25 mg g-1 FW in the Si-treated samples. In Garganega, Si treatment was correlated with increased proline levels. In Chardonnay, the Si application was related to decreased tartaric acid. The results of this work show for the first time that Si induces cultivar specific changes in the berry composition in plants cultivated without an evident abiotic or biotic stress.

Advances in Plant Sulfur Research.

As an essential nutrient required for plant growth and development, sulfur (S) deficiency in productive systems limits yield and quality. This special issue hosts a collection of original research articles, mainly based on contributions from the 11th International Plant Sulfur Workshop held on 16-20 September 2018 in Conegliano, Italy, focusing on the following topics: (1) The germinative and post-germinative behaviour of Brassica napus seeds when severe S limitation is applied to the parent plants; (2) the independence of S deficiency from the mRNA degradation initiation enzyme PARN in Arabidopsis; (3) the glucosinolate distribution in the aerial parts of sel1-10, a disruption mutant of the sulfate transporter SULTR1;2, in mature Arabidopsis thaliana plants; (4) the accumulation of S-methylcysteine as its γ-glutamyl dipeptide in Phaseolus vulgaris; and (5) the role of ferric iron chelation-strategy components in the leaves and roots of maize, have provided new insights into the effect of S availability on plant functionality. Moreover, the role of S deficiency in root system functionality has been highlighted, focusing on (6) the contribution of root hair development to sulfate uptake in Arabidopsis, and (7) the modulation of lateral root development by the CLE-CLAVATA1 signaling pathway under S deficiency. The role of S in plants grown under drought conditions has been investigated in more detail focusing (8) on the relationship between S-induced stomata closure and the canonical ABA signal transduction machinery. Furthermore, (9) the assessment of S deficiency under field conditions by single measurements of sulfur, chloride, and phosphorus in mature leaves, (10) the effect of fertilizers enriched with elemental S on durum wheat yield, and (11,12) the impact of elemental S on the rhizospheric bacteria of durum wheat contributed to enhance the scientific knowledge on S nutrition under field conditions.

Sulfur metabolic engineering enhances cadmium stress tolerance and root to shoot iron translocation in Brassica napus L.

Serine acetyltransferase (SAT) (EC 2.3.1.30) is the rate-limiting enzyme of cysteine (Cys) biosynthesis, providing the decisive precursor for the ubiquitous defense thiol glutathione (GSH). Together with O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) SAT generates Cys in the cytosol, plastids, and mitochondria of vascular plants. The current study aimed to overproduce Cys and GSH for enhanced stress tolerance via overexpression of the feedback-insensitive isoform of serine acetyltransferase from tobacco, i.e., NtSAT4. Constitutive overexpression of NtSAT4 in Brassica napus resulted in the 2.6-fold-4-fold higher SAT activity in different subcellular compartment-specific lines. This higher SAT activity led to a 2.5-fold-3.5-fold higher steady-state level of free Cys and 2.2-fold-5.3-fold elevated level of GSH in leaves compared with nontransformed plants. Among the compartment-specific lines, the mitochondrial targeted NtSAT4 overexpressor line M-182 showed the highest levels of Cys (3.5-fold) and GSH (5.3-fold) compared with wild-type plants. Overexpression of NtSAT4 conferred a physiological advantage in terms of enhanced tolerance against oxidative stress with hydrogen peroxide and the heavy metal cadmium (Cd). The NtSAT4 overexpressor lines showed a significantly higher amount of iron (Fe) translocation from roots to shoots compared with nontransformed plants. Overall, these results suggest that overexpression of NtSAT4 is a promising approach to creating plants with tolerance to heavy metals and oxidative stress and, in addition, may potentially improve plant nutrition in terms of enhanced Fe translocation from roots to shoots.

Sulfate-Induced Stomata Closure Requires the Canonical ABA Signal Transduction Machinery.

Phytohormone abscisic acid (ABA) is the canonical trigger for stomatal closure upon abiotic stresses like drought. Soil-drying is known to facilitate root-to-shoot transport of sulfate. Remarkably, sulfate and sulfide-a downstream product of sulfate assimilation-have been independently shown to promote stomatal closure. For induction of stomatal closure, sulfate must be incorporated into cysteine, which triggers ABA biosynthesis by transcriptional activation of NCED3. Here, we apply reverse genetics to unravel if the canonical ABA signal transduction machinery is required for sulfate-induced stomata closure, and if cysteine biosynthesis is also mandatory for the induction of stomatal closure by the gasotransmitter sulfide. We provide genetic evidence for the importance of reactive oxygen species (ROS) production by the plasma membrane-localized NADPH oxidases, RBOHD, and RBOHF, during the sulfate-induced stomatal closure. In agreement with the established role of ROS as the second messenger of ABA-signaling, the SnRK2-type kinase OST1 and the protein phosphatase ABI1 are essential for sulfate-induced stomata closure. Finally, we show that sulfide fails to close stomata in a cysteine-biosynthesis depleted mutant. Our data support the hypothesis that the two mobile signals, sulfate and sulfide, induce stomatal closure by stimulating cysteine synthesis to trigger ABA production.

Interactions between chromium and sulfur metabolism in Brassica juncea.

The effects of chromate on sulfate uptake and assimilation were investigated in the accumulator Brassica juncea (L.) Czern. Seven-day-old plants were grown for 2 d under the following combination of sulfate and chromate concentration: (i) no sulfate and no chromate (-S), (ii) no sulfate and 0.2 mmol L(-1) chromate (-S +Cr), (iii) 1 mmol L(-1) sulfate and no chromate (+S), or (iv) 1 mmol L(-1) sulfate and 0.2 mmol L(-1) chromate (+S +Cr). Despite the toxic effects exerted by chromate as indicated by altered level of reducing sugars and proteins in leaves, the growth of B. juncea was only weakly reduced by chromate, and no variation in chlorophyll a and b was measured, regardless of S availability. Chromium (Cr) was stored more in roots than in leaves, and the maximum Cr accumulation was measured in -S +Cr plants. The significant decrease of the sulfate uptake rates observed in Cr-treated plants was accompanied by a repression of the root low-affinity sulfate transporter (BjST1), suggesting that the transport of chromate in B. juncea may involve sulfate carriers. Once absorbed, chromate induced genes involved in sulfate assimilation (ATP-sulfurylase: atps6; APS-reductase: apsr2; Glutathione synthethase: gsh2) and accumulation of cysteine and glutathione, which may suggest that these reduced S compounds play a role in Cr tolerance. Together, our findings indicate that when phytoremediation technologies are used to recover Cr-contaminated areas, the concentration of sulfate in the plant growth medium must be considered because it may influence the ability of plants to accumulate and tolerate Cr.

Fragmentation of the main triterpene acids of apple by LC-APCI-MSn.

In this paper, we investigated the fragmentation of the main triterpene acids of apple using an liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MSn ) approach and high-resolution mass spectrometry (HR-MS) (Q-TOF). Triterpenes were isolated using semipreparative high-performance liquid chromatography, and chemical structures were elucidated by HR-MS and nuclear magnetic resonance spectroscopy. Finally, compounds were used to study MSn behavior in ion trap. Isolated triterpenes present similar structures, bearing carboxyl group linked to C-17 and different substitutions. We observed significant changes in MS2 spectra, which were useful for further compound identification. The observed fragments allowed the discrimination of different derivatives, namely, pomaceic, annurcoic, euscaphic, pomolic, corosolic, maslinic, betulinic, oleanolic, and ursolic acids. The proposed method allows a rapid identification of triterpene acids, and it could be useful for the analysis of these compounds in apple fruits and in other natural sources.

Phytoremediation of chromium using Salix species: cloning ESTs and candidate genes involved in the Cr response.

In this research a differential display based on the detection of cDNA-AFLP markers was used to identify candidate genes potentially involved in the regulation of the response to chromium in four different willow species (Salix alba, Salix eleagnos, Salix fragilis and Salix matsudana) chosen on the basis of their suitability in phytoremediation techniques. Our approach enabled the assay of a large set of mRNA-related fragments and increased the reliability of amplification-based transcriptome analysis. The vast majority of transcript-derived fragments were shared among samples within species and thus attributable to constitutively expressed genes. However, a number of differentially expressed mRNAs were scored in each species and a total of 68 transcripts displaying an altered expression in response to Cr were isolated and sequenced. Public database querying revealed that 44.1% and 4.4% of the cloned ESTs score significant similarity with genes encoding proteins having known or putative function, or with genes coding for unknown proteins, respectively, whereas the remaining 51.5% did not retrieve any homology. Semi-quantitative RT-PCR analysis of seven candidate genes fully confirmed the expression patterns obtained by cDNA-AFLP. Our results indicate the existence of common mechanisms of gene regulation in response to Cr, pathogen attack and senescence-mediated programmed cell death, and suggest a role for the genes isolated in the cross-talk of the signaling pathways governing the adaptation to biotic and abiotic stresses.

Different leachate phytotreatment systems using sunflowers.

The use of energy crops in the treatment of wastewaters is of increasing interest, particularly in view of the widespread scarcity of water in many countries and the possibility of obtaining renewable fuels of vegetable origin. The aim of this study was to evaluate the feasibility of landfill leachate phytotreatment using sunflowers, particularly as seeds from this crop are suitable for use in biodiesel production. Two different irrigation systems were tested: vertical flow and horizontal subsurface flow, with or without effluent recirculation. Plants were grown in 130L rectangular tanks placed in a special climatic chamber. Leachate irrigated units were submitted to increasing nitrogen concentrations up to 372mgN/L. Leachate was successfully tested as an alternative fertilizer for plants and was not found to inhibit biomass development. The experiment revealed good removal efficiencies for COD (η>50%) up until flowering, while phosphorous removal invariably exceeded 60%. Nitrogen removal rates decreased over time in all experimental units, particularly in vertical flow tanks. In general, horizontal flow units showed the best performances in terms of contaminant removal capacity; the effluent recirculation procedure did not improve performance. Significant evapo-transpiration was observed, particularly in vertical flow units, promoting removal of up to 80% of the inlet irrigation volume.