1,721,032 research outputs found
IMPACT OF PHENYLPROPANOIDS ON HEAT STRESS PROTECTION
No full textI metaboliti secondari sono composti organici non direttamente coinvolti nella crescita e nello sviluppo della pianta. Queste sostanze sono spesso prodotte e accumulate dopo stress biotici e abiotici, ma le loro precise funzioni in vivo sono ancora poco chiare. Le colture cellulari in vitro possono essere utilizzate al fine di chiarire il ruolo biologico di specifici metaboliti secondari dopo un particolare tipo di stress. In una precedente tesi di dottorato, uno stress termico di 1 ora a 44°C è stato applicato ad una linea cellulare di carota chiamata R3M, una linea pigmentata, in grado di accumulare derivati della cianidina e degli acidi idrossicinnamici, molecole appartenenti alla classe dei fenilpropanoidi. Il trattamento termico causava la comparsa di strutture circolari citoplasmatiche dopo 24 ore di recupero dallo stress, ed è stato dimostrato che le cellule mostranti queste strutture erano destinate ad una lenta morte cellulare, con caratteristiche tipiche di una morte cellulare programmata. Somministrando alle cellule R3M alcuni precursori molecolari (approccio chimico), quali gli acidi idrossicinnamici, prima del trattamento termico, si è osservata una riduzione del numero di cellule con strutture circolari citoplasmatiche e una riduzione del numero di cellule morte. Questo progetto ha come obiettivo la caratterizzazione dei danni morfologici causati dallo stress termico, al fine di chiarire il possibile ruolo biologico di specifici fenilpropanoidi accumulati nella coltura R3M. Inoltre, un tentativo per valutare la possibilità di sfruttare l'approccio chimico, ovvero di somministrare i precursori molecolari in pianta per osservare l’eventuale assorbimento e modificazione degli stessi in piante intere, è stato provato. La doppia colorazione con fluoresceina di acetato e ER tracker blue white delle cellule stressate al calore, ha permesso di investigare la natura di queste strutture circolari citoplasmatiche, rivelando che esse sono circondate dal reticolo endoplasmatico. Questa organizzazione è tipica degli autofagosomi, strutture coinvolte nel processo di macroautofagia e spesso indotte da uno stress. Ulteriori analisi di microscopia hanno mostrato come queste strutture circolari citoplasmatiche contengano anche gocce lipidiche e organelli, per esempio cromoplasti, supportando fortemente l'ipotesi che queste strutture circolari siano autofagosomi. La colorazione con il marcante di endocitosi “FM 1-43” ha permesso di osservare che, nelle cellule stressate, l'arresto dei movimenti endocellulari avveniva immediatamente dopo l’esposizione allo stress termico e che, anche dopo due ore di recupero, la ripresa dei movimenti vescicolari non avveniva. Questo risultato ha suggerito che il citoscheletro, o qualche componente implicato nel trasporto vescicolare, poteva essere stato danneggiato dal calore. Dato che il citoscheletro è formato da diverse strutture, due tossine, colchicina e citocalasina D, agenti contro i microtubuli e microfilamenti, sono state addizionate alla coltura cellulare di carota per provare a mimare i danni causati dallo stress termico. Solo la citocalasina D perfettamente fenocopiava, in una parte della popolazione cellulare, la morfologia stressata, suggerendo che uno dei target dello stress termico erano i microfilamenti, i quali, una volta danneggiati, determinavano, direttamente o indirettamente, la comparsa di putativi autofagosomi nelle cellule stressate. Poiché l'approccio di somministrazione degli acidi idrossicinnamici determinava l'aumento di specifiche antocianine e derivati di acidi idrossicinnamici, prevenendo parzialmente la comparsa di putativi autofagosomi nelle cellule stressate, è stato concluso che questi fenilpropanoidi agiscono contro lo stress termico, prevenendo la morte cellulare programmata, probabilmente mediante un effetto protettivo, non caratterizzato, sui microfilamenti. Rispetto a molte pubblicazioni scientifiche che suggeriscono il ruolo dei metaboliti secondari sulla base di una correlazione tra accumulo/diminuzione di gruppi/classi di metaboliti e uno specifico stress, questo lavoro dimostra chiaramente che alcune molecole specifiche, in particolare la cianidina acilata con acido caffeico e l’acido cumarico coniugato con l’acido quinico, hanno un ruolo biologico di protezione contro lo stress termico in questa linea cellulare carota, risultando in una diminuzione della morte cellulare. Infine, la somministrazione di precursori molecolari è stata condotta su piante intere, rivelando che le piantine di Arabidopsis thaliana sono in grado di assorbire precursori molecolari e convertirli in altri prodotti. Questo risultato dimostra che l’approccio chimico può essere eseguito non solo in colture cellulari in vitro, ma anche in piante intere, permettendo future indagini sui ruoli biologici dei metaboliti secondari.Secondary metabolites are organic compounds not directly involved in growth and development. These substances are often produced by plants after biotic and abiotic stresses, but their precise in vivo functions are still unclear. In vitro cell culture can be used in order to clarify the role of certain secondary metabolites after stress. In a previous work, a heat stress (1 hour at 44°C) was applied to R3M carrot cell line, a purple pigmented line that is able to accumulate cyanidin derivatives and hydroxycinnamic acid derivatives, molecules belonging to the phenylpropanoid class. The heat treatment caused the appearance of cytoplasmic circular structures after 24 hours of recovery. It has been shown that cells developing these structures were committed to a slow cell death, which showed some morphological markers of a programmed cell death. Feeding R3M cells with hydroxycinnamic acids before the heat treatment caused a reduction of the number of cells with cytoplasm circular structures and a reduction of cell death. The aim of this project is the characterization of the morphological damages caused by heat stress in order to clarify the possible biological role of specific phenylpropanoids accumulated in R3M cells. Finally, an effort to evaluate the possibility to exploit the feeding chemical approach in in vitro whole plants has been pursuit. The double staining of the heat stressed cells with fluorescein di-acetate and ER tracker blue-white allowed at observing the appearance of cytoplasmic circular structures surrounded by endoplasmic reticulum. This organization is typical of autophagosomes, structures involved during the macroautophagy process, which are often stress induced. Further microscope investigations revealed that these circular structures included lipid droplets and also organelles, for instance chromoplasts, strongly supporting that these structures are autophagosomes. The staining of the heat stressed cells with the endocytosis tracker FM 1-43 allowed to observe the arrest of endocellular movements just after heat stress, suggesting that cytoskeleton could have been damaged by heat. Toxins towards microtubules and microfilaments were used in order to phenocopy the damages induced by the heat treatment, but only Cytochalasin D, an anti-microfilament agent, caused the formation of structures similar to the heat induced putative-autophagosomes. Since the feeding approach determined the increase of anthocyanins and hydroxycinnamic acid derivatives, thus partially preventing the putative autophagosome-containing cell phenotype after heat stress, it can be concluded that these phenylpropanoids prevent programmed cell death possibly through an uncharacterized protective effect on microfilaments. Respect to other public researches that show the correlations between groups or classes of molecules and stresses, this work clearly demonstrates that specific molecules, i.e. the cyanidin acylated with caffeic acid and coumaroyl quinic acid, have a protective role against heat stress in this carrot cell line. Finally, the administration of molecular precursors has been investigated on in vitro whole plants revealing that Arabidopsis thaliana seedlings are able to absorb molecular precursors and immediately convert them in other plant products. This finding demonstrates that the chemical approach can be performed also in whole plants, allowing future investigations on secondary metabolite biological roles
Temperature and solar irradiation effects on secondary metabolism during ripening of field-grown everbearing strawberries
No full textThe garden strawberry (Fragaria x ananassa Duch.) is cultivated and consumed worldwide because of the pleasant flavor and health-promoting phytochemicals of its false fruits. Monocrop cultivars produce fully ripe strawberries in about one month post-anthesis throughout the spring, while everbearing cultivars undergo additional strawberry production in autumn. In this work, we evaluated the impact of different season-dependent environmental conditions on the ripening program of an everbearing field-gown strawberry variety from autumn 2015 to spring 2016. We combined ad hoc sampling and environmental data collection with LC-MS-based untargeted metabolomics to dissect the effects of cumulative temperature and solar irradiation on fruit quality parameters and secondary metabolism during ripening. Different dynamics in specific sub-groups of metabolites were observed in strawberries experiencing distinct amounts of cumulative temperature and solar irradiation during spring and autumn. The integration of statistical analyses on collected data revealed that solar irradiation mainly affected fruit fresh weight and organic acid levels, whereas temperature had a more selective effect on the accumulation of specific flavonols, anthocyanins, and soluble sugar. These findings are of suitable interest to design further approaches for the study of the complex interactions among environmental conditions and ripening in strawberries grown in a real-world scenario
Performance comparison of electrospray ionization and atmospheric pressure chemical ionization in untargeted and targeted liquid chromatography/mass spectrometry based metabolomics analysis of grapeberry metabolites
Full text linkElectrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are both used to generate ions for the analysis of metabolites by liquid chromatography/mass spectrometry (LC/MS). We compared the performance of these methods for the analysis of Corvina grapevine berry methanolic extracts, which are complex mixtures of diverse metabolites
Untargeted Metabolomics Analysis of the Orchid Species Oncidium sotoanum Reveals the Presence of Rare Bioactive C-Diglycosylated Chrysin Derivatives
Full text linkPlants are valuable sources of secondary metabolites with pharmaceutical properties, but only a small proportion of plant life has been actively exploited for medicinal purposes to date. Underexplored plant species are therefore likely to contain novel bioactive compounds. In this study, we investigated the content of secondary metabolites in the flowers, leaves and pseudobulbs of the orchid Oncidium sotoanum using an untargeted metabolomics approach. We observed the strong accumulation of C-diglycosylated chrysin derivatives, which are rarely found in nature. Further characterization revealed evidence of antioxidant activity (FRAP and DPPH assays) and potential activity against neurodegenerative disorders (MAO-B inhibition assay) depending on the specific molecular structure of the metabolites. Natural product bioprospecting in underexplored plant species based on untargeted metabolomics can therefore help to identify novel chemical structures with diverse pharmaceutical properties
Metabolite Profiling Reveals Developmental Inequalities in Pinot Noir Berry Tissues Late in Ripening
Full text linkUneven ripening in Vitis vinifera is increasingly recognized as a phenomenon of interest, with substantial implications for fruit and wine composition and quality. This study sought to determine whether variation late in ripening (∼Modified Eichhorn-Lorenz stage 39) was associated with developmental differences that were observable as fruits within a cluster initiated ripening (véraison). Four developmentally distinct ripening classes of berries were tagged at cluster véraison, sampled at three times late in ripening, and subjected to untargeted HPLC-MS to measure variation in amino acids, sugars, organic acids, and phenolic metabolites in skin, pulp, and seed tissues separately. Variability was described using predominantly two strategies. In the first, multivariate analysis (Orthogonal Projections to Latent Structures-Discriminant Analysis, OPLS-DA) was used to determine whether fruits were still distinguishable per their developmental position at véraison and to identify which metabolites accounted for these distinctions. The same technique was used to assess changes in each tissue over time. In a second strategy and for each annotated metabolite, the variance across the ripening classes at each time point was measured to show whether intra-cluster variance (ICV) was growing, shrinking, or constant over the period observed. Indeed, berries could be segregated by OPLS-DA late in ripening based on their developmental position at véraison, though the four ripening classes were aggregated into two larger ripening groups. Further, not all tissues were dynamic over the period examined. Although pulp tissues could be segregated by time sampled, this was not true for seed and only moderately so for skin. Ripening group differences in seed and skin, rather than the time fruit was sampled, were better able to define berries. Metabolites also experienced significant reductions in ICV between single pairs of time points, but never across the entire experiment. Metabolites often exhibited a combination of ICV expansion, contraction and persistence. Finally, we observed significant differences in the abundance of some metabolites between ripening classes that suggest the berries that initiated ripening first remained developmentally ahead of the lagging fruit even late in the ripening phase. This presents a challenge to producers who would seek to harvest at uniformity or at a predefined level of variation
Advances in combined enzymatic extraction of ferulic acid from wheat bran
No full textWheat bran could be utilised as feedstock for innovative and sustainable biorefinery processes. Here, an enzymatic hydrolysis process for ferulic acid (FA) extraction was optimised step by step for total wheat bran (Tritello) and then also applied to the outer bran layer (Bran 1). Proteins, reducing sugars, total phenols and FA were quantified. The highest FA yields (0.82–1.05 g/kg bran) were obtained either by rehydrating the bran by autoclaving (Tritello) or by steam explosion (Bran 1) using a bran/water ratio of 1:20, followed by enzymatic pretreatment with Alcalase and Termamyl, to remove protein and sugars, and a final enzymatic hydrolysis with Pentopan and feruloyl esterase to solubilise phenol. FA was recovered from the final digestate via solid phase extraction. A 40-fold scale-up was also performed and the release of compounds along all the process steps and at increasing incubation times was monitored. Results showed that FA was initially present at a minimum level while it was specifically released during the enzymatic treatment. In the final optimized process, the FA extraction yield was higher than that obtained with NaOH control hydrolysis while, in comparison with other FA enzymatic extraction methods, fewer process steps were required and no buffers, strong acid/alkali nor toxic compounds were used. Furthermore, the proposed process may be easily scaled-up, confirming the feasibility of wheat bran valorisation by biorefinery processes to obtain valuable compounds having several areas of potential industrial exploitation
Investigating the Chemobiodiversity of Bryophytes through Untargeted Metabolomics
No full textAmong the large and diversified plant species that populate our planet, bryophytes certainly occupy an important place in the plant kingdom. This group of ancient non-vascular plants evolved more than 350 million years ago from green algae, and comprise more than 20.000 species, considering Mosses, Hornworts and Liverworts. Bryophytes have adapted to live in different environments by developing several adaptations and strategies during their evolution, including the biosynthesis of specialized metabolites. However, information about the metabolome composition of most Bryophyte species remains limited. This work focuses on the characterization of the metabolic profiles of a range of Bryophyte species, including mosses and liverworts. It is part of a larger project, the National Biodiversity Future Center, which aims to explore the chemo-biodiversity of species belonging to Italian flora to discover metabolites exerting possible biological activities towards human health and plant growth promotion and protection. Regarding mosses, the dried plants were initially hydrated and sampled after 7 days. The vegetative material was cleaned from soil particles and dead parts, and subsequently sampled forming 3 different representative biological replicates. For liverworts, plants were immediately cleaned and sampled. Plants were frozen with liquid nitrogen, homogenized to a fine powder and extract with methanol. To elucidate the metabolic profile of the collected species, the methanol extracts were diluted and analysed by following an untargeted metabolomics approach with a UPLC-HRMS technique. Samples were also analyzed in FAST-DDA mode (data-dependent analysis) to assist the subsequent identification analysis, which is actually ongoing
Untargeted metabolomics: an emerging approach to determine the composition of herbal products
Full text linkNatural remedies, such as those based on traditional Chinese medicines, have become more popular also in western countries over the last 10 years. The composition of these herbal products is largely unknown and difficult to determine. Moreover, since plants respond to their environment changing the metabolome, the composition of plant material can vary depending on the plant growth conditions.However, there is a growing need of a deeper knowledge on such natural remedies also in view of the growing number of reports of toxicity following the consumption of herbal supplements. Untargeted metabolomics is a useful approach for the simultaneous analysis of many compounds in herbal products. In particular, liquid chromatography/mass spectrometry (LC-MS) can determine presence, amount and sometime structures of plant metabolites in complex herbal mixtures, with significant advantages over techniques such as nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC-MS)
Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes
Full text linkPlant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required
La modificazione del profilo dei fenilpropanoidi influenza la sensibilità allo stress termico di cellule di Daucus carota
No full textLe piante sono in grado di produrre un vasto e diverso assortimento di metaboliti secondari a seguito di stress biotici e abiotici, ma le precise funzioni in vivo di queste molecole non sono ancora state delucidate. Una possibile strategia per chiarire il ruolo di specifici metaboliti secondari consiste nel modulare il profilo metabolico di linee cellulari in vitro e valutarne l’effetto sulla risposta a stress. La coltura cellulare di Daucus carota R3M, una linea pigmentata selezionata nel nostro laboratorio, presenta un caratteristico profilo metabolico ricco di antocianine e derivati degli acidi idrossicinnamici. Mediante somministrazione di precursori e/o inibitori della via metabolica dei fenilpropanoidi, si è modificato il livello di specifici metaboliti secondari nelle cellule, verificando l’effetto dei trattamenti mediante analisi metabolomica degli estratti metabolici attraverso HPLC-DAD e HPLC-ESI-MS/MS. Come stress abiotico si è utilizzato uno stress termico di 1h a 44°C. L’effetto di tale stress è stato valutato dal punto di vista citologico, mediante microscopia ottica e confocale. Dal punto di vista citologico, il trattamento termico causa gravi danni alle cellule di carota, attivando un processo di morte lenta (possibile PCD), preceduta da un rallentamento dei movimenti intracellulari, da una riarrangiamento delle briglie citoplasmatiche dall’aggregazione del citoplasma in “placche” circolari interne, simili a vacuoli autofagici, oltre a un danno ossidativo alle membrane cellulari. Al fine di verificare se danni al citoscheletro potessero essere coinvolti negli effetti citologici dello stress termico, sono state somministrate le cellule, in assenza di stress, tossine contro alcuni elementi del citoscheletro. Si è visto che la somministrazione di citocalasina D, ad opportune concentrazioni, mima l’effetto citologico dello stress termico, suggerendo che lo stress termico determini danni e microfilamenti, causando la formazione delle “placche” citoplasmatiche che anticipano la morte cellulare programmata. Si è quindi modificato il profilo metabolico delle cellule e l'effetto di queste modificazioni sulla risposta allo stress termico è stato valutato comparando la percentuale di cellule con fenotipo stressato, nei campioni sottoposti a stress termico, pre-trattati o meno che vari precursori. La somministrazione di diidroquercetina (precursore delle antocianine) ed i singoli acidi idrossicinnamici determina l’incremento dell’accumulo di specifici fenilpropanoidi, tra cui antocianine glicosilate e acilate e acidi idrossicinnamici liberi e una diminuzione del numero di cellule con fenotipo stressato. La somministrazione dell’acido piperonilico, inibitore della biosintesi dell’acido cumarico (intermedio nella biosintesi delle antocianine e substrato della loro acilazione) determina una diminuzione del livello di specifici fenilpropanoidi e un aumento del numero di cellule con fenotipo stressato. Questi dati suggeriscono che specifici fenilpropanoidi possano avere un effetto protettivo dallo stress termico, anche se l’analisi dettagliata della correlazione fra livello di metaboliti E protezione da stress è tuttora in corso
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