Andrea Occhipinti

Supercritical Fluid Extraction of Plant Flavors and Fragrances

Supercritical fluid extraction (SFE) of plant material with solvents like CO2, propane, butane, or ethylene is a topic of growing interest. SFE allows the processing of plant material at low temperatures, hence limiting thermal degradation, and avoids the use of toxic solvents. Although today SFE is mainly used for decaffeination of coffee and tea as well as production of hop extracts on a large scale, there is also a growing interest in this extraction method for other industrial applications operating at different scales. In this review we update the literature data on SFE technology, with particular reference to flavors and fragrance, by comparing traditional extraction techniques of some industrial medicinal and aromatic crops with SFE. Moreover, we describe the biological activity of SFE extracts by describing their insecticidal, acaricidal, antimycotic, antimicrobial, cytotoxic and antioxidant properties. Finally, we discuss the process modelling, mass-transfer mechanisms, kinetics parameters and thermodynamic by giving an overview of SFE potential in the flavors and fragrances arena.

UV-B modulates the interplay between terpenoids and flavonoids in peppermint (Mentha × piperita L.).

Modulation of secondary metabolites by UV-B involves changes in gene expression, enzyme activity and accumulation of defence metabolites. After exposing peppermint (Mentha x piperita L.) plants grown in field (FP) and in a growth chamber (GCP) to UV-B irradiation, we analysed by qRT-PCR the expression of genes involved in terpenoid biosynthesis and encoding: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (Mds), isopentenyl diphosphate isomerase (Ippi), geranyl diphosphate synthase (Gpps), (-)-limonene synthase (Ls), (-)-limonene-3-hydroxylase (L3oh), (+)-pulegone reductase (Pr), (-)-menthone reductase (Mr), (+)-menthofuran synthase (Mfs), farnesyl diphosphate synthase (Fpps) and a putative sesquiterpene synthase (S-TPS). GCP always showed a higher terpenoid content with respect to FP. We found that in both FP and GCP, most of these genes were regulated by the UV-B treatment. The amount of most of the essential oil components, which were analysed by gas chromatography-mass spectrometry (GC-MS), was not correlated to gene expression. The total phenol composition was found to be always increased after UV-B irradiation; however, FP always showed a higher phenol content with respect to GCP. Liquid chromatography-mass spectrometry (LC-ESI-MS/MS) analyses revealed the presence of UV-B absorbing flavonoids such as eriocitrin, hesperidin, and kaempferol 7-O-rutinoside whose content significantly increased in UV-B irradiated FP, when compared to GCP. The results of this work show that UV-B irradiation differentially modulates the expression of genes involved in peppermint essential oil biogenesis and the content of UV-B absorbing flavonoids. Plants grown in field were better adapted to increasing UV-B irradiation than plants cultivated in growth chambers. The interplay between terpenoid and phenylpropanoid metabolism is also discussed.

Ginkgo biloba Responds to Herbivory by Activating Early Signaling and Direct Defenses

Background Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses. Methodology/Principal Findings Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca2+]cyt) and H2O2 production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca2+]cyt and H2O2. Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides. Conclusions/Significance By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this “living fossil” plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function.

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K(+) channel activity, cytosolic calcium [Ca2+]cyt and reactive oxygen species (ROS) (H2 O2 and NO) release, shoot-to-root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative β-galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca2+]cyt , NO and H2 O2 . In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K(+) channel activity and Vm depolarization, a loss of shoot-induced root-Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium-activated oxidative response and a K(+) -dependent Vm-activated jasmonate response associated with the release of VOCs.

Dynamics of Membrane Potential Variation and Gene Expression Induced by Spodoptera littoralis, Myzus persicae, and Pseudomonas syringae in Arabidopsis

Background Biotic stress induced by various herbivores and pathogens invokes plant responses involving different defense mechanisms. However, we do not know whether different biotic stresses share a common response or which signaling pathways are involved in responses to different biotic stresses. We investigated the common and specific responses of Arabidopsis thaliana to three biotic stress agents: Spodoptera littoralis, Myzus persicae, and the pathogen Pseudomonas syringae. Methodology/Principal Findings We used electrophysiology to determine the plasma membrane potential (Vm) and we performed a gene microarray transcriptome analysis on Arabidopsis upon either herbivory or bacterial infection. Vm depolarization was induced by insect attack; however, the response was much more rapid to S. littoralis (30 min −2 h) than to M. persicae (4–6 h). M. persicae differentially regulated almost 10-fold more genes than by S. littoralis with an opposite regulation. M. persicae modulated genes involved in flavonoid, fatty acid, hormone, drug transport and chitin metabolism. S. littoralis regulated responses to heat, transcription and ion transport. The latest Vm depolarization (16 h) was found for P. syringae. The pathogen regulated responses to salicylate, jasmonate and to microorganisms. Despite this late response, the number of genes differentially regulated by P. syringae was closer to those regulated by S. littoralis than by M. persicae. Conclusions/Significance Arabidopsis plasma membranes respond with a Vm depolarization at times depending on the nature of biotic attack which allow setting a time point for comparative genome-wide analysis. A clear relationship between Vm depolarization and gene expression was found. At Vm depolarization timing, M. persicae regulates a wider array of Arabidopsis genes with a clear and distinct regulation than S. littoralis. An almost completely opposite regulation was observed between the aphid and the pathogen, with the former suppressing and the latter activating Arabidopsis defense responses.

Chrysolina herbacea modulates terpenoid biosynthesis of Mentha aquatica L.

Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plants response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insects response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase.

Magnetoreception: an unavoidable step for plant evolution?

The geomagnetic field (GMF) is steadily acting on living systems, and influences many biological processes. In animals, the mechanistic origin of the GMF effect has been clarified and cryptochrome has been suggested as a chemical magnetoreceptor. Here we propose a possible role for the GMF variations in plant evolution.

Differential expression of CPKs and cytosolic Ca2+ variation in resistant and susceptible apple cultivars (Malus x domestica) in response to the pathogen Erwinia amylovora and mechanical wounding

BackgroundPlant calcium (Ca2+) signals are involved in a wide array of intracellular signalling pathways following pathogen invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate signalling following Ca2+ influx after pathogen infection. However, to date this prediction has remained elusive.ResultsWe conducted a genome-wide identification of the Malus x domestica CPK (MdCPK) gene family and identified 30 CPK genes. Comparative phylogenetic analysis of Malus CPKs with CPKs of Arabidopsis thaliana (AtCPKs), Oryza sativa (OsCPKs), Populous trichocarpa (PtCPKs) and Zea mays (ZmCPKs) revealed four different groups. From the phylogenetic tree, we found that MdCPKs are closely related to AtCPKs and PtCPKs rather than OsCPKs and ZmCPKs, indicating their dicot-specific origin. Furthermore, comparative quantitative real time PCR and intracellular cytosolic calcium ([Ca2+]cyt) analysis were carried out on fire blight resistant and susceptible M. x domestica apple cultivars following infection with a pathogen (Erwinia amylovora) and/or mechanical damage. Calcium analysis showed an increased [Ca2+]cyt over time in resistant cultivars as compared to susceptible cultivars. Gene expression studies showed that 11 out of the 30 MdCPKs were differentially expressed following pathogen infection.ConclusionsWe studied the genome-wide analysis of MdCPK gene family in Malus x domestica and analyzed their differential gene expression along with cytosolic calcium variation upon pathogen infection. There was a striking difference in MdCPKs gene expressions and [Ca2+]cyt variations between resistant and susceptible M. x domestica cultivars in response to E. amylovora and mechanical wounding. Our genomic and bioinformatic analysis provided an important insight about the role of MdCPKs in modulating defence responses in susceptible and resistant apple cultivars. It also provided further information on early signalling and downstream signalling cascades in response to pathogenic and mechanical stress.

Chemical partitioning and antioxidant capacity of green coffee (Coffea arabica and Coffea canephora) of different geographical origin.

Green coffee beans of Coffea arabica and Coffea canephora accessions from different geographical origin (Brazil, Colombia, Ethiopia, Honduras, Kenya, Mexico, Peru, Uganda and Vietnam) were extracted and the extracts analyzed by HPLC-ESI-MS/MS for the identification and quantification of chlorogenic acids and caffeine content. Principal component and cluster analyses were used to identify chemical patterns separating the different species and accessions based on their geographical origin. C. canephora showed always a higher caffeine content with respect to C. arabica, whereas the C. arabica accessions from Kenya showed a higher chlorogenic acids and a lower caffeine content. The antioxidant capacity of green coffee extracts was assayed by the reducing power and DPPH assays. The antioxidant capacity correlated with the chlorogenic acids content. The results show that the C. arabica from Kenya possesses the highest chlorogenic acids/caffeine ratio and, among the C. arabica accessions, the highest antioxidant capacity. Therefore, the C. arabica from Kenya is the most suitable green coffee source for nutraceutical applications because of its high antioxidant capacity and low caffeine content.

Plant defences against ants provide a pathway to social parasitism in butterflies

Understanding the chemical cues and gene expressions that mediate herbivore–host-plant and parasite–host interactions can elucidate the ecological costs and benefits accruing to different partners in tight-knit community modules, and may reveal unexpected complexities. We investigated the exploitation of sequential hosts by the phytophagous–predaceous butterfly Maculinea arion, whose larvae initially feed on Origanum vulgare flowerheads before switching to parasitize Myrmica ant colonies for their main period of growth. Gravid female butterflies were attracted to Origanum plants that emitted high levels of the monoterpenoid volatile carvacrol, a condition that occurred when ants disturbed their roots: we also found that Origanum expressed four genes involved in monoterpene formation when ants were present, accompanied by a significant induction of jasmonates. When exposed to carvacrol, Myrmica workers upregulated five genes whose products bind and detoxify this biocide, and their colonies were more tolerant of it than other common ant genera, consistent with an observed ability to occupy the competitor-free spaces surrounding Origanum. A cost is potential colony destruction by Ma. arion, which in turn may benefit infested Origanum plants by relieving their roots of further damage. Our results suggest a new pathway, whereby social parasites can detect successive resources by employing plant volatiles to simultaneously select their initial plant food and a suitable sequential host.