Impact of Phenylpropanoid Compounds on Heat Stress Tolerance in Carrot Cell Cultures.

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated in vitro, focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on projection to latent structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton.

Phytochemical analysis of Passiflora loefgrenii Vitta, a rich source of luteolin-derived flavonoids with antioxidant properties.

OBJECTIVES: The paper describes the flavonoid composition of the aerial parts (young leaves, YL; adult leaves, AL; stems, ST) of Passiflora loefgrenii Vitta, a rare species native to Brazil, where it is traditionally used as food. Antioxidant potential has also been evaluated. To the best of our knowledge, no phytochemical and biological study on this species has been reported previously. METHODS: Compositional data have been acquired combining HPLC-diode array detector (DAD) and Electrospary ionization-tandem mass spectrometry (ESI-MS/MS) analyses. Antioxidant activity has been evaluated by the 2,2'-di-phenyl-1-picrylhydrazyl method. KEY FINDINGS: Glycosylated flavones, with luteolin as the main aglycone, can be regarded as biomarkers for this drug. Qualitative composition of the extracts from YL, AL and ST was similar. The bulk of the constituents was made up by 8-C-ß-glucosyl luteolin (orientin), 7-O-α-rhamnosyl-6-C-ß-glucosyl luteolin and 6-C-α-rhamnosyl luteolin, which totally amounted to 16.57 (73%), 10.77 (74%) and 5.07 (77%) µg/mg in YL, AL and ST, respectively. P. loefgrenii showed a good antioxidant activity (IC50 of 350 µg/ml), higher than generally reported for other passifloras. CONCLUSIONS: P. loefgrenii, rich in luteolin glycosides, can be regarded as a good candidate to be explored for therapeutic properties other than the sedative one since it represents a rich source of valuable flavonoids with antioxidant potential.

Carrot-specific features of the phenylpropanoid pathway identified by feeding cultured cells with defined intermediates.

Plants produce a vast array of secondary metabolites, many of which have important biological properties in animals when consumed as part of the diet. Interestingly, although the activities and benefits of plant secondary metabolites in animals are well established, comparatively little is known about the endogenous functions of these compounds in plants. One way to investigate the role of secondary products in plants is to modify the secondary metabolome and investigate the impact of such modifications on the phenotype. We have designed a novel feeding approach using different hydroxycinnamic acids (HCAs) and the cyanidin precursor dihydroquercetin (DHQ) to modify the metabolome of carrot R3M suspension cells. This strategy increased the accumulation of specific metabolites in a predictable way, and provided novel insights into the carrot phenylpropanoid pathway, suggesting that (a) cells use HCA hexose esters as substrates in the biosynthetic pathway leading to the accumulation of the various HCA derivatives and (b) p-coumaric acid derivative levels play a key roles in the regulation the flux of HCAs along the pathway. Moreover, this rapid strategy for metabolome modification does not depend on the availability of molecular tools or knowledge and can therefore be applied to any plant species.

Metabolomic analysis reveals that the accumulation of specific secondary metabolites in Echinacea angustifolia cells cultured in vitro can be controlled by light.

Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14 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.

Correlated accumulation of anthocyanins and rosmarinic acid in mechanically stressed red cell suspensions of basil (Ocimum basilicum).

A red basil cell line (T2b) rich in rosmarinic acid (RA) was selected for the stable production of anthocyanins (ACs) in the dark. Cell suspension cultures were subjected to mechanical stress through increased agitation (switch from 90 to 150 rpm) to determine the relationship between AC and RA accumulation. Cell extracts were analyzed by HPLC and LC-MS, and the resulting data were processed with multivariate statistical analysis. MS and MS/MS spectra facilitated the putative annotation of several complex cyanidin-based ACs, which were esterified with coumaric acid and, in some cases, also with malonic acid. It was also possible to identify various RA-related molecules, some caffeic and coumaric acid derivatives and some flavanones. Mechanical stress increased the total AC and RA contents, but reduced biomass accumulation. Many metabolites were induced by mechanical stress, including RA and some of its derivatives, most ACs, hydroxycinnamic acids and flavonoids, whereas the abundance of some RA dimers was reduced. Although AC and RA share a common early biosynthetic pathway (from phenylalanine to 4-coumaroyl-CoA) and could have similar or overlapping functions providing antioxidant activity against stress-generated reactive oxygen species, there appeared to be no competition between their individual pathways.

Metabolomics of Daucus carota cultured cell lines under stressing conditions reveals interactions between phenolic compounds.

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.

Pre-analytical method for metabolic profiling of plant cell cultures of Passiflora garckei.

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.

Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota.

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. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.

Reduction of cell size induced by enod40 in Arabidopsis thaliana.

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.