Cinzia M. Bertea

Gain and Loss of Fruit Flavor Compounds Produced by Wild and Cultivated Strawberry Species

The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as α-pinene and β-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of α-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of α-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H2O2) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H2O2 was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H2O2 was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H2O2 in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H2O2 prompted a transient transmembrane potential (Vm) depolarization, with a Vm depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca2+-sensing aequorin system, increasing amounts of added H2O2 correlated with a higher cytosolic calcium ([Ca2+]cyt) concentration. In MD and HW leaves, H2O2 also triggered the increase of [Ca2+]cyt, but MD-elicited [Ca2+]cyt increase was more pronounced when compared to HW leaves after addition of exogenous H2O2. The results clearly indicate that Vm depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

BackgroundPlant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable.ResultsTo investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.ConclusionsThese results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

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.

UV-A effects on photomorphogenesis and essential-oil composition in Mentha piperita

Abstract UV-A radiation (360 nm) affects the photomorphogenesis and essential-oil chemical composition of Mentha piperita. Total phenols and chlorophyll b are also affected by UV-A. When UV-A is given during the day (UV-A+W), a significant increase is found in total leaf area, leaf-area index, chlorophyll a/b ratio, total phenols, total essential-oil content, menthofuran and menthol. UV-A irradiation during the night period induces a typical shade-avoidance syndrome, with stem elongation, decreased leaf area and leaf-area index, decreased protein and total phenol content, and decreased essential-oil and menthol content. The possible interference of UV-A with phytochrome is discussed.

Effect of (+)-pulegone and other oil components of Mentha x Piperita on cucumber respiration.

Peppermint (Mentha x piperita L.) essential oil and main components were assessed for their ability to interfere with plant respiratory functions. Tests were conducted on both root segments and mitochondria isolated by etiolated seedlings of cucumber (Cucumis sativus L.). Total essential oil inhibited 50% of root and mitochondrial respiration (IC50) when used at 324 and 593 ppm, respectively. (+)-Pulegone was the most toxic compound, with a 0.08 and 0.12 mM IC50 for root and mitochondrial respiration, respectively. (-)-Menthone. followed (+)-pulegone in its inhibitory action (IC50 values of 1.11 and 2.30 mM for root and mitochondrial respiration respectively), whereas (-)-menthol was the less inhibitory compound (IC50 values of 1.85 and 3.80 mM respectively). A positive correlation was found for (+)-pulegone, (-)-menthone and (-)-menthol between water solubility and respiratory inhibition. The uncoupling agent. carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP), lowered (-)-menthol and (-)menthone inhibition and annulled (+)-pulegone inhibition of mitochondrial respiration, whereas salicyl-hydroxamic acid (SHAM) 2-hydroxybenzohydroxamic acid, the alternative oxidase (AO) inhibitor, increased (-)-menthone inhibition and annulled both (+)-pulegone and (-)-menthol inhibitory activity. The possible interaction of (-)-pulegone and (-)-menthol with AO and the mechanism of action of(+)-pulegone, (-)-menthone and (-)-menthol on mitochondrial respiration are discussed.

Effect of benzoic acid hydroxy- and methoxy- ring substituents during cucumber (Cucumis sativus L.) germination. I.: Isocitrate lyase and catalase activity

Abstract Benzoic acids (BAs) are released by plants through leaching of leaf exudates and rhizodeposition. Most of them exert allelopathic effects. BAOH ring substituents generally caused an increase in germination percentage, whereas OCH 3 ring substituents decreased the percentage of germination. Hydroxy substituents increased isocitrate lyase (ICL) activity, whereas OCH 3 ring substituents decreased enzyme activity indicating a direct effect on enzyme catalysis. The addition of BAs to the reaction mixture containing extracts from seedlings incubated with the related BA derivatives and to extracts from controls confirmed the direct stimulating (OH) and inhibiting (OCH 3 ) effect of BA substituents on ICL catalysis. Catalase (CAT) activity was dramatically increased by hydroxylated BAs, even though some methoxylated BAs exerted a strong activating effect. Native and SDS-PAGE analyses indicate the presence of two bands of 236 and 66 kDa, which correspond to ICL enzyme activity levels. The enhancing of silver staining and the disappearance of these bands are related to ICL enzyme activity suggesting a possible effect of BAs on synthesis of ICL. The physiological effect of BAs on cucumber germination is also discussed.

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.