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Chemical characterization of plant materials and development of analytical methodologies for metabolite determination
No full textPlants synthesize a huge number of metabolites, involved in essential life functions (primary metabolites) or in particular defense, signaling and development roles (secondary metabolites). Plant metabolome remains for a large part uncharacterized, due to the high variability among species. The investigations of this rich variety of plant metabolites have been improved in the 20th century with the development of new analytical techniques, which permitted the identification of low-abundant molecules and their structural characterization. In this study we present different approaches for the characterization of the chemical composition of plant materials. Taking advantage from the use of HRMS technology, we developed both quantitative and qualitative methods, in order to obtain a comprehensive profiling of metabolites. In the first research chapter we describe a study of wild and transgenic Nicotiana Langsdorfii plants, exposed to different abiotic stresses. These plants have been traditionally used for genetic and physiologic studies. The plants were modified by the insertion of the Rol C gene, from Agrobacterium rhizogenes, and of the rat glucocorticoid receptor (GR). The aim of this study was the investigation of the metabolic changes associated with the genetic modifications and stress exposition, in order to highlight eventual advantages deriving from the inserted genes and to better understand the effects of abiotic stresses. In the second study we considered two different species of the Glycyrrhiza genus, G. Glabra and G. Uralensis, commonly used for the production of licorice. The characterization of the two G. Glabra varieties, glandulifera and typica, was also carried out. The application of the metabolomic method aimed to the characterization of the chemical composition of each species, which is directly involved in the quality of the derived products. The third study report the analysis of the fruits of Coffea Canephora and Coffea Arabica, the most cultivated species for the production of coffee beans. The different parts of the fruits (perisperm, endosperm, pulp) were collected separately in two different harvest seasons. The metabolomic analysis was performed by integration of two analytical approaches: the use of HPLC-HRMS for the detection of secondary metabolites and the use of GC-TOF for the identification of primary metabolites.
This work showed the potential of the integration of different approaches in analytical chemistry, contributing to the comprehension of plant stress response and suggesting some possible application of the genetic modifications tested. Moreover we provide useful information about licorice and coffee, demonstrating the potential of metabolomic methodology as a tool in the food characterization and quality assurance. The metabolomic analyses also permit to suggest a few biosynthetic pathway regulations involved in stress response and ripening process. The study represents a good starting point for future works in the field of foodomics and of system biology, highlighting original findings, not reported before, which should be better investigated
Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses
Get PDFThe presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgene
Processes affecting the distribution of PCBs in the Southern Ocean
No full textPolychlorinated biphenyls (PCBs) are a broad class of globally distributed persistent pollutants that differ in their degree of chlorination and, thereby, in their volatility and are subject to long-range atmospheric transport (LRAT). Although their industrial production was discontinued in the early nineties, some PCB congeners are still released into the environment as unintentional by-products of dye manufacturing and other chemical productions. Among them, 3,3’-dichlorobiphenyl (PCB-11) has been detected at concentrations often higher than those of the other technical congeners (i.e. legacy Aroclor PCBs) in almost all the environmental compartments, even in polar areas (Choi et al., 2008).
It is known that the atmosphere plays a key role in transport and distribution of persistent organic pollutants (POPs) towards polar areas, through successive phases of volatilization and deposition that occur at the air/water interface. The low temperatures of the polar areas promote this partition between the atmosphere and the seawater surface via the cold trapping mechanism (Wania and MacKay, 1996). However, this process is reversible, so the partition of POPs moves in one direction or in the opposite one depending on the volatility of the molecules involved, their relative concentration in air and water, and changes in temperature (Galbán-Malagón et al., 2013).
As the surface water temperature decreases, lighter congeners, more prone to volatilization, tend instead to settle in the water surface layers. Indeed, in the Southern Ocean, where the temperature of surface water is reduced to values close to that of the air, a particularly high concentration of PCB-11 compared to that of other less volatile PCB congeners have been reported (Choi et al., 2008; Pizzini et al., 2017). Instead, PCBs with a higher degree of chlorination are less prone to volatilization in temperate areas and, consequently, they would be preferentially transferred to the Southern Ocean through Modified Circumpolar Deep waters (Fuoco et al., 2009) rather than via LRAT. Against this background, it can be assumed that more processes are involved in the transport of PCBs towards the Southern Ocean, depending on the characteristics of the investigated molecules and, primarily, their volatility.
In this work, the results of analyses of water samples collected along a transect from the Southern Pacific Ocean to the Ross Sea will be presented. Preliminary outcomes confirmed the hypothesis that more volatile PCBs reach the Southern Ocean preferentially through a cold condensation process, differently from heavier ones. Di- and Tri-chlorinated PCBs reached particularly high concentrations in water surface layers where there is a sharp decrease in temperature, in the Antarctic convergence zone, while this effect is much more limited for the less volatile investigated congeners.
Choi, S.-D., Baek, S.-Y., Chang, Y.-S., Wania, F., Ikonomou, M.G., Yoon, Y.-J., Park, B.-K., Hong, S., 2008. Passive Air Sampling of Polychlorinated Biphenyls and Organochlorine Pesticides at the Korean Arctic and Antarctic Research Stations: Implications for Long-Range Transport and Local Pollution. Environmental Science & Technology 42, 7125-7131. https://doi.org/10.1021/es801004p.
Fuoco, R., Giannarelli, S., Wei, Y., Ceccarini, A., Abete, C., Francesconi, S., Termine, M., 2009. Persistent organic pollutants (POPs) at Ross Sea (Antarctica). Microchemical Journal 92(1), 44-48. https://doi.org/10.1016/j.microc.2008.11.004.
Galbán-Malagón, C. J., Del Vento, S., Cabrerizo, A., Dachs, J., 2013. Factors affecting the atmospheric occurrence and deposition of polychlorinated biphenyls in the Southern Ocean. Atmospheric Chemistry and Physics 13, 12029-12041. https://doi.org/10.5194/acp-13-12029-2013.
Pizzini, S., Sbicego, C., Corami, F., Grotti, M., Magi, E., Bonato, T., Cozzi, G., Barbante, C., Piazza, R., 2017. 3,3’-dichlorobiphenyl (non-Aroclor PCB-11) as a marker of non-legacy PCB contamination in marine species: comparison between Antarctic and Mediterranean bivalves. Chemosphere 175, 28-35. https://doi.org/10.1016/j.chemosphere.2017.02.023.
Wania, F., MacKay, D., 1996. Tracking the Distribution of Persistent Organic Pollutants. Environmental Science & Technology 30(9), 390A-396A. https://doi.org/10.1021/es962399q
A new exploration of licorice metabolome
No full textThe roots and rhizomes of licorice plants (genus Glycyrrhiza L.) are commercially employed, after processing, in confectionery production or as sweetening and flavouring agents in the food, tobacco and beer industries. G. glabra, G. inflata and G. uralensis are the most significant licorice species, often indistinctly used for different productions. Licorice properties are directly related to its chemical composition, which determines the commercial values and the quality of the derived products. In order to better understand the characteristics and properties of each species, a chemical characterization of three species of licorice (G. glabra, G. inflata, G. uralensis) is proposed, through an untargeted metabolomic approach and using high-resolution mass spectrometry. The statistical analysis reveals new possible markers for the analyzed species, and provides a reliable identification of a high number of metabolites, contributing to the characterization of Glycyrrhiza metabolome
Nicotiana langsdorffiiwild type and genetically modified exposed to chemical and physical stress: changes in element content
No full textThe concentrations of 19 elements in wild and genetically modified Nicotiana langsdorffii
(N. langsdorffii) exposed to Chromium (VI) and to water deficit were determined and
compared to provide new information about their response to abiotic stress.
Genetic modifications by GR and RolC genes (encoding for the rat glucocorticoid receptor
and for Agrobacterium rhizogenes RolC, respectively) were investigated because they induce
significant, but only partially known changes in the plant response to stress.
Simultaneous determination of Al, As, Ba, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, Mg, Mn, Na, Pb,
Rb, Sr, V and Zn was carried out by ICP-MS equipped with a collision/reaction cell (ICPORS-
MS). The methodology was optimised by testing the grinding, homogenisation, digestion
and analysis procedures, to reduce the uncertainty of the experimental results and to
identify statistically significant differences between nine sample pools, for a total of 75
samples. The quality control procedure was carried out by blank control and by evaluating
the detection limits and repeatability. Trueness was assessed by analysing certified reference
material, NIST 1573a.
Significant differences were observed in the uptake and accumulation of several elements
in the wild-type N. langsdorffii samples, either with respect to the plants submitted to water
deficit and exposure to Cr(VI) or with respect to the genetically modified plants. The
differences were highlighted by principal component analysis (PCA). The analysis of the
element content of the whole plant, combined with the data found in the literature, allows us
to hypothesise effects on the metabolic mechanism controlling the uptake and translocation of
elements inside the vegetal organism. Because genetic and chemical stress decreases the
nutrient concentration in the whole plant, we can say that the uptake at root level is affected.
The increase in concentration of elements such as As, Sr and Al indicates a decreased
selectivity in the uptake of potentially toxic elements and, consequently, highlights the effects
on the plant’s metabolic processes
Effects of Water Deficit and Heat Stress on Nicotiana langsdorffii Metabolomic Pattern Modified by Insertion of rolD Gene from Agrobacterium rhizogenes
Get PDFAbiotic stresses are major factors that negatively affect plant growth and productivity. Plants have developed complex strategies to ensure their survival and reproduction under adverse conditions, activating mechanisms that involve changes at different metabolic levels. In order to select stress-resistant species, research has focused on molecular studies and genetic engineering, showing promising results. In this work, the insertion of the rolD gene from Agrobacterium rhizogenes into Nicotiana langsdorffii plants is investigated, in order to assess the potential of this genetic modification towards mitigating water and heat stresses. Different approaches were combined: a high-throughput metabolomics and ionomics study was performed, together with the determination of important plant phytohormones. The aim was to identify the influence of abiotic stresses on plants and to highlight the effects of the rolD genetic modification on plant stress response. The most relevant compounds for each kind of stress were identified, belonging mainly to the classes of lipids, acyl sugars, glycosides, and amino acid derivatives. Water stress (WS) determined a decrease of elements and secondary metabolites, while amino acids and their derivatives increased, proving to be key molecules in this type of stress. RolD plants exposed to high temperature stress (HS) presented higher dry weight levels than controls, as well as increased amounts of K and adenosine and lower levels of damage-associated metabolites, suggesting the increased resistance of rolD-modified plants toward HS.Abiotic stresses are major factors that negatively affect plant growth and productivity. Plants have developed complex strategies to ensure their survival and reproduction under adverse conditions, activating mechanisms that involve changes at different metabolic levels. In order to select stress-resistant species, research has focused on molecular studies and genetic engineering, showing promising results. In this work, the insertion of therolDgene fromAgrobacterium rhizogenesintoNicotiana langsdorffiiplants is investigated, in order to assess the potential of this genetic modification towards mitigating water and heat stresses. Different approaches were combined: a high-throughput metabolomics and ionomics study was performed, together with the determination of important plant phytohormones. The aim was to identify the influence of abiotic stresses on plants and to highlight the effects of therolDgenetic modification on plant stress response. The most relevant compounds for each kind of stress were identified, belonging mainly to the classes of lipids, acyl sugars, glycosides, and amino acid derivatives. Water stress (WS) determined a decrease of elements and secondary metabolites, while amino acids and their derivatives increased, proving to be key molecules in this type of stress. RolD plants exposed to high temperature stress (HS) presented higher dry weight levels than controls, as well as increased amounts of K and adenosine and lower levels of damage-associated metabolites, suggesting the increased resistance ofrolD-modified plants toward HS
Ionomic responses of wild and transgenic nicotiana langsdorffii plants exposed to heat stress
No full textPlants are composed of chemical elements, which uptake, distribution and accumulation are controlled by genetic and environmental factors. Particularly, plants face adverse or limiting conditions by activating a complex system of physiological and metabolic responses. High temperatures, together with other stress factors induced by global climate change, may significantly affect the growth and development of plants; heat stress conditions showed to delay plant’s growth, damage its cell membranes, increase transpiration and reduce the opening of the stomata. In this work, Nicotiana langsdorffii plants, wild and transgenic for the Agrobacterium rhizogenes rol C and rol D gene and the rat glucocorticoid receptor (GR) gene, were exposed to heat stress. The responses of the transgenic plants to the abiotic stresses were assessed by the analysis of the plant ionomic profile, the metabolomic data and the phytormonal profile (1,2). Simultaneous determination of 12 elements was carried out by ICP-MS equipped with a collision/reaction cell (ICP-ORS-MS). WT, Rol D and Rol C plants showed higher changes both in the ionomic and in the metabolic profile, suggesting that these genotypes could be more affected by heat stress exposition. On the contrary GR plants showed almost unchanged values of many elements and organic compounds (including phytohormone and lipids) indicating that this genetic modification could provide a higher plant resistance toward heat stress. Statistical analysis permitted to highlight the main significant differences among the metabolic responses of the examined genotype
Effect of different rootstocks on element uptake
No full textThe research results of the last decades shown that the industrial and the inadequate agricultural activity may increase the concentration of potentially toxic elements in soil.
The grape rootstock is a filter system in the grapevine, which prevent the accumulation in the grape berry of some trace elements of high concentrations from the soil.
Therefore it is very important, in the wine sector, to understand which rootstock should be applied, that uptake the toxic elements of the soil only in low concentration, in order to reduce the final absorption in the must.
The choice of the rootstock is very important, not only as regards the up-take of potentially harmful elements, but also because the nature and characteristics of a rootstock are able to influence the organoleptic properties of wine. Rootstock varieties differently affect fruiting, rate of growth, yield and fruit quality [1].
In this research 60 germoplasm accessions including different rootstocks commonly used (eg. Kober 5 BB, 1103 Paulsen, 110 Richter), other hybrids rarely used (eg. 216-3 Castel, 1616 Couderc ) and Vitis species used as parental to form hybrid rootstocks (eg. V. cinerea, V. berlandieri) were studied. The concentration of some trace elements and nutrients were examined in leaves rootstocks to define the different metal up-take mechanisms, verifying before the homogeneity of land cultivation.
Simultaneous determination of Al, As, Ba, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, Mg, Mn, Na, Pb, Rb, Sr, V and Zn was carried out by ICP-MS equipped with a collision/reaction cell (ICPORS-MS). The methodology was optimised by testing the grinding, homogenisation, digestion and analysis procedures, to reduce the uncertainty of the experimental results as in a previous study [2]
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