ABSTRACT The genus Passiflora comprises hundred species, mainly native of the South American trop... more ABSTRACT The genus Passiflora comprises hundred species, mainly native of the South American tropics and rainforests, which are grouped into 21 subgenera. Some species are widely studied for their economic importance and are chiefly cultivated for production of fruit juice. To obtain a continuous source of material for a screening of secondary metabolites, zygotic embryo culture was attempted for 62 Passiflora species, starting from seeds mainly collected in the wild. Twenty nine of these species produced calli, which had very different growth rates. Plants were successfully regenerated from calli of 13 different species. For 25 of the responsive species this is the first report of in vitro culture.
A metabolomic approach followed by principal components and partial least square analysis was use... more A metabolomic approach followed by principal components and partial least square analysis was used for investigating the effect of environmental factors on two Daucus carota L. cv. Flakkese cell lines (R3M and R4G), selected for their ability to produce anthocyanins in the light and the dark, respectively. A positive correlation between total anthocyanin, hydroxycinnamic and hydroxybenzoic acid accumulation was found in both lines. Furthermore, the experimental design and the combination of biochemical and statistical analyses allowed us to unravel complex relationships between environmental factors and phenylpropanoid composition. Among these, the induction by mechanical stress of overproduction of all anthocyanins, hydroxycinnamic and hydroxybenzoic acids except sinapic acid derivatives, whose accumulation was inhibited.
Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but... more Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14Â days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography-mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds.
Microscopic and flow cytometric analysis of protoplasts derived from carrot (Daucus carota L.) ce... more Microscopic and flow cytometric analysis of protoplasts derived from carrot (Daucus carota L.) cells in culture revealed two different cellular subpopulations that differ in their light-scattering properties and in the emission of red autofluorescence. The two subpopulations were sorted on the basis of scattering properties and cultured independently. Subpopulation 1, characterized by higher side scattering (SS) values, had a higher
... of meristematic pea root tissue for biochemical and flow cytometry applications Donato Chiata... more ... of meristematic pea root tissue for biochemical and flow cytometry applications Donato Chiatante, Paola Brusa, Marisa Levi, Sergio Sgorbati and Elio Sparvoli ... This hole had a rim to hold a rubberring. Two Miracloth layers, held in place by the rubber ring, closed the hole. ...
Page 1. PHYSIOL. PLANT. 51: 321-325. Copenhagen 1981 Lack of correlation between overall protein ... more Page 1. PHYSIOL. PLANT. 51: 321-325. Copenhagen 1981 Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis Maria Grazia Galli, Marisa Levi and Elio Sparvoli ...
An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model ... more An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level.
We established a step-by-step, experimentguided metabolomics procedure, based on LC-ESI-MS analys... more We established a step-by-step, experimentguided metabolomics procedure, based on LC-ESI-MS analysis, to generate a detailed picture of the changing metabolic profiles during late berry development in the important Italian grapevine cultivar Corvina. We sampled berries from four developmental time points and three postharvest time points during the withering process, and used chromatograms of methanolic extracts to test the performance of the MetAlign and MZmine data mining programs. MZmine achieved a better resolution and therefore generated a more useful data matrix. Then both the quantitative performance of the analytical platform and the matrix effect were assessed, and the final dataset was investigated by multivariate data analysis. Our analysis confirmed the results of previous studies but also revealed some novel findings, including the prevalence of two specific flavonoids in unripe berries and important differences between the developmental profiles of flavones and flavanones, suggesting that specific individual metabolites could have different functions, and that flavones and flavanones probably play quite distinct biological roles. Moreover, the hypothesis-free multivariate analysis of subsets of the wide data matrix evidentiated the relationships between the various classes of metabolites, such as those between anthocyanins and hydroxycinnamic acids and between flavan-3-ols and anthocyanins.
nuclei, and to map DNA sequences on chromosomes. Mapping resolution is higher on pachytene chromo... more nuclei, and to map DNA sequences on chromosomes. Mapping resolution is higher on pachytene chromosomes Fluorescence in situ hybridization (FISH) is a wellthan on metaphase chromosomes (Shen et al., 1987; established technique used for the detection of Zhong et al., 1996), due to their less contracted structure; specific DNA regions, that has been applied to it is even higher in interphase nuclei (Lawrence et al., interphase nuclei, pachytene and metaphase chromo-1988, 1990 Trask et al., 1989 Trask et al., , 1991. In the latter case, somes as well as to extended DNA fibres. This techhowever, the possibility of identifying individual chromonique allows the physical mapping of specific DNA somes is lost. The use of FISH on extended DNA fibres sequences both on individual chromosomes and further enhances the physical mapping resolution (de extended fibres. A new FISH protocol is described here Jong et al., 1999), due to the high linear stretching degrees that enhances the sensitivity of the method. Probes of chromatin, evaluated to be around 3.27 kb mm−1, for small unique DNA sequences of less than 2 kb give according to Fransz et al. (Fransz et al., 1996a). high signal-to-noise ratio with this method, and can
In Vitro Cellular & Developmental Biology - Plant, 1993
We used cultured pea embryo axes to study the effect of exogenous abscisic acid (ABA) on the reac... more We used cultured pea embryo axes to study the effect of exogenous abscisic acid (ABA) on the reactivation of cell proliferation at germination. ABA (100 ttM) was administered in a complete medium or in a medium without KNO 3 (the main source of K + in the complete medium). In the presence of ABA, only a few meristematic root tip nuclei progressed through the cell cycle during the first 24 h of germination. Compared to the control the percentage of Go/G 1 cells remained high. In the absence of KN03, the inhibitory effect of ABA on the cell cycle increased and the transition from G1 to S was almost completely inhibited after 24 h of germination. Moreover, the amount of a 47 kDa nuclear protein remained high in embryos treated with ABA whereas it decreased in the control.
In Vitro Cellular & Developmental Biology - Plant, 1992
A method for culturing excised pea embryo axes, which allows growth of large batches of embryo ax... more A method for culturing excised pea embryo axes, which allows growth of large batches of embryo axes in a reduced volume of medium and is suitable for performing biochemical analyses and early treatment or labeling of embryos with exogenous substances, has been devised. Pea embryo axes were excised from the seeds after 3 h of imbibition and cultured in a mineral medium containing sucrose, vitamins, and casamino acids. Under these conditions, the reactivation of the cell cycle occurs much earlier than in intact seeds: with flow cytometry and bromodeoxyuridine labeling, cells in the S phase were found in the root meristems after only 12 h from the beginning of imbibition. The transition of cells from a quiescent to a proliferative state is accompanied by changes in the electrophoretic pattern of nuclear proteins, particularly with regard to two proteins of apparent molecular weight of 90 and 46 kDa, as shown previously in normally germinating seeds.
Methanolic extracts were prepared from different organs of plants from five Passiflora species ob... more Methanolic extracts were prepared from different organs of plants from five Passiflora species obtained by zygotic embryo culture and evaluated for their capacity to quench DPPH and ABTS + radicals in comparison to that of Trolox, a water soluble vitamin-E analogue. Moreover their antimicrobial activity against E. coli was tested by agar diffusion and turbidity assays. P. nitida, P. foetida, and P. palmeri showed antimicrobial activity. P. nitida and P. palmeri also showed high antioxidant activity. P. tenuifila and P. coriacea demonstrated antioxidant power but not antimicrobial activity. The phenolic content of the different extracts was studied and quantified by spectrophotometric methods, HPLC, and mass spectrometry. High antioxidant activity correlated with high amounts of o-diphenol and catechin. An unknown component, tentatively identified as structural isomer of isoschaftoside, appeared to correlate with antimicrobial activity.
Passiflora garckei cell cultures were used as a model to describe a reproducible sample preparati... more Passiflora garckei cell cultures were used as a model to describe a reproducible sample preparation method. Solid phase extraction (SPE) was employed to isolate the plant metabolites for nuclear magnetic resonance (NMR) analysis and to subsequently detect the differences between yeast extract elicited and control cells. Compared with previous results obtained by using a Sephadex LH-20 column, SPE coupled with NMR spectroscopy improves the analysis of aromatic compounds e.g.: trans-feruloyl derivatives and trans-coumaroyl derivatives. Moreover, it decreases the concentration of sugars that usually overlap with many plant metabolite signals.
An extensive analysis of organ and cell size was performed in three different Arabidopsis lines t... more An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.
ABSTRACT The genus Passiflora comprises hundred species, mainly native of the South American trop... more ABSTRACT The genus Passiflora comprises hundred species, mainly native of the South American tropics and rainforests, which are grouped into 21 subgenera. Some species are widely studied for their economic importance and are chiefly cultivated for production of fruit juice. To obtain a continuous source of material for a screening of secondary metabolites, zygotic embryo culture was attempted for 62 Passiflora species, starting from seeds mainly collected in the wild. Twenty nine of these species produced calli, which had very different growth rates. Plants were successfully regenerated from calli of 13 different species. For 25 of the responsive species this is the first report of in vitro culture.
A metabolomic approach followed by principal components and partial least square analysis was use... more A metabolomic approach followed by principal components and partial least square analysis was used for investigating the effect of environmental factors on two Daucus carota L. cv. Flakkese cell lines (R3M and R4G), selected for their ability to produce anthocyanins in the light and the dark, respectively. A positive correlation between total anthocyanin, hydroxycinnamic and hydroxybenzoic acid accumulation was found in both lines. Furthermore, the experimental design and the combination of biochemical and statistical analyses allowed us to unravel complex relationships between environmental factors and phenylpropanoid composition. Among these, the induction by mechanical stress of overproduction of all anthocyanins, hydroxycinnamic and hydroxybenzoic acids except sinapic acid derivatives, whose accumulation was inhibited.
Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but... more Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14Â days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography-mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds.
Microscopic and flow cytometric analysis of protoplasts derived from carrot (Daucus carota L.) ce... more Microscopic and flow cytometric analysis of protoplasts derived from carrot (Daucus carota L.) cells in culture revealed two different cellular subpopulations that differ in their light-scattering properties and in the emission of red autofluorescence. The two subpopulations were sorted on the basis of scattering properties and cultured independently. Subpopulation 1, characterized by higher side scattering (SS) values, had a higher
... of meristematic pea root tissue for biochemical and flow cytometry applications Donato Chiata... more ... of meristematic pea root tissue for biochemical and flow cytometry applications Donato Chiatante, Paola Brusa, Marisa Levi, Sergio Sgorbati and Elio Sparvoli ... This hole had a rim to hold a rubberring. Two Miracloth layers, held in place by the rubber ring, closed the hole. ...
Page 1. PHYSIOL. PLANT. 51: 321-325. Copenhagen 1981 Lack of correlation between overall protein ... more Page 1. PHYSIOL. PLANT. 51: 321-325. Copenhagen 1981 Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis Maria Grazia Galli, Marisa Levi and Elio Sparvoli ...
An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model ... more An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level.
We established a step-by-step, experimentguided metabolomics procedure, based on LC-ESI-MS analys... more We established a step-by-step, experimentguided metabolomics procedure, based on LC-ESI-MS analysis, to generate a detailed picture of the changing metabolic profiles during late berry development in the important Italian grapevine cultivar Corvina. We sampled berries from four developmental time points and three postharvest time points during the withering process, and used chromatograms of methanolic extracts to test the performance of the MetAlign and MZmine data mining programs. MZmine achieved a better resolution and therefore generated a more useful data matrix. Then both the quantitative performance of the analytical platform and the matrix effect were assessed, and the final dataset was investigated by multivariate data analysis. Our analysis confirmed the results of previous studies but also revealed some novel findings, including the prevalence of two specific flavonoids in unripe berries and important differences between the developmental profiles of flavones and flavanones, suggesting that specific individual metabolites could have different functions, and that flavones and flavanones probably play quite distinct biological roles. Moreover, the hypothesis-free multivariate analysis of subsets of the wide data matrix evidentiated the relationships between the various classes of metabolites, such as those between anthocyanins and hydroxycinnamic acids and between flavan-3-ols and anthocyanins.
nuclei, and to map DNA sequences on chromosomes. Mapping resolution is higher on pachytene chromo... more nuclei, and to map DNA sequences on chromosomes. Mapping resolution is higher on pachytene chromosomes Fluorescence in situ hybridization (FISH) is a wellthan on metaphase chromosomes (Shen et al., 1987; established technique used for the detection of Zhong et al., 1996), due to their less contracted structure; specific DNA regions, that has been applied to it is even higher in interphase nuclei (Lawrence et al., interphase nuclei, pachytene and metaphase chromo-1988, 1990 Trask et al., 1989 Trask et al., , 1991. In the latter case, somes as well as to extended DNA fibres. This techhowever, the possibility of identifying individual chromonique allows the physical mapping of specific DNA somes is lost. The use of FISH on extended DNA fibres sequences both on individual chromosomes and further enhances the physical mapping resolution (de extended fibres. A new FISH protocol is described here Jong et al., 1999), due to the high linear stretching degrees that enhances the sensitivity of the method. Probes of chromatin, evaluated to be around 3.27 kb mm−1, for small unique DNA sequences of less than 2 kb give according to Fransz et al. (Fransz et al., 1996a). high signal-to-noise ratio with this method, and can
In Vitro Cellular & Developmental Biology - Plant, 1993
We used cultured pea embryo axes to study the effect of exogenous abscisic acid (ABA) on the reac... more We used cultured pea embryo axes to study the effect of exogenous abscisic acid (ABA) on the reactivation of cell proliferation at germination. ABA (100 ttM) was administered in a complete medium or in a medium without KNO 3 (the main source of K + in the complete medium). In the presence of ABA, only a few meristematic root tip nuclei progressed through the cell cycle during the first 24 h of germination. Compared to the control the percentage of Go/G 1 cells remained high. In the absence of KN03, the inhibitory effect of ABA on the cell cycle increased and the transition from G1 to S was almost completely inhibited after 24 h of germination. Moreover, the amount of a 47 kDa nuclear protein remained high in embryos treated with ABA whereas it decreased in the control.
In Vitro Cellular & Developmental Biology - Plant, 1992
A method for culturing excised pea embryo axes, which allows growth of large batches of embryo ax... more A method for culturing excised pea embryo axes, which allows growth of large batches of embryo axes in a reduced volume of medium and is suitable for performing biochemical analyses and early treatment or labeling of embryos with exogenous substances, has been devised. Pea embryo axes were excised from the seeds after 3 h of imbibition and cultured in a mineral medium containing sucrose, vitamins, and casamino acids. Under these conditions, the reactivation of the cell cycle occurs much earlier than in intact seeds: with flow cytometry and bromodeoxyuridine labeling, cells in the S phase were found in the root meristems after only 12 h from the beginning of imbibition. The transition of cells from a quiescent to a proliferative state is accompanied by changes in the electrophoretic pattern of nuclear proteins, particularly with regard to two proteins of apparent molecular weight of 90 and 46 kDa, as shown previously in normally germinating seeds.
Methanolic extracts were prepared from different organs of plants from five Passiflora species ob... more Methanolic extracts were prepared from different organs of plants from five Passiflora species obtained by zygotic embryo culture and evaluated for their capacity to quench DPPH and ABTS + radicals in comparison to that of Trolox, a water soluble vitamin-E analogue. Moreover their antimicrobial activity against E. coli was tested by agar diffusion and turbidity assays. P. nitida, P. foetida, and P. palmeri showed antimicrobial activity. P. nitida and P. palmeri also showed high antioxidant activity. P. tenuifila and P. coriacea demonstrated antioxidant power but not antimicrobial activity. The phenolic content of the different extracts was studied and quantified by spectrophotometric methods, HPLC, and mass spectrometry. High antioxidant activity correlated with high amounts of o-diphenol and catechin. An unknown component, tentatively identified as structural isomer of isoschaftoside, appeared to correlate with antimicrobial activity.
Passiflora garckei cell cultures were used as a model to describe a reproducible sample preparati... more Passiflora garckei cell cultures were used as a model to describe a reproducible sample preparation method. Solid phase extraction (SPE) was employed to isolate the plant metabolites for nuclear magnetic resonance (NMR) analysis and to subsequently detect the differences between yeast extract elicited and control cells. Compared with previous results obtained by using a Sephadex LH-20 column, SPE coupled with NMR spectroscopy improves the analysis of aromatic compounds e.g.: trans-feruloyl derivatives and trans-coumaroyl derivatives. Moreover, it decreases the concentration of sugars that usually overlap with many plant metabolite signals.
An extensive analysis of organ and cell size was performed in three different Arabidopsis lines t... more An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.
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