The dominant allele Aft induces a shift from flavonol to anthocyanin production in response to UV-B radiation in tomato fruit

The introgression of the A ft allele into domesticated tomato induced a shift from flavonol to anthocyanin production in response to UV-B radiation, while the hp - 1 allele negatively influenced the response of flavonoid biosynthesis to UV-B. Introgression of the dominant allele Anthocyanin fruit (Aft) from Solanum chilense induces anthocyanin accumulation in the peel of tomato (Solanum lycopersicum L.) fruit. UV-B radiation can influence plant secondary metabolism regulating the expression of several genes, among which those involved in flavonoid biosynthesis. Here, we investigated whether post-harvest UV-B treatment could up-regulate flavonoid production in tomato fruits and whether the Aft allele could affect flavonoid biosynthesis under UV-B radiation. Mature green fruits of an anthocyanin-rich tomato mutant line (SA206) and of its wild-type reference, cv. Roma, were daily subjected to post-harvest UV-B treatment until full ripening. Up-regulation of CHS and CHI transcription by UV-B treatment induced flavonoid accumulation in the peel of cv. Roma. Conversely, UV-B decreased the total flavonoid content and CHS transcript levels in the SA206 peel. SA206 being a double mutant containing also hp-1 allele, we investigated also the behavior of hp-1 fruit. The decreased peel flavonoid accumulation and gene transcription in response to UV-B suggest that hp-1 allele is involved in the marked down-regulation of the flavonoid biosynthesis observed in SA206 fruit. Interestingly, in SA206, UV-B radiation promoted the synthesis of delphinidin, petunidin, and malvidin by increasing F3'5'H and DFR transcription, but it decreased rutin production, suggesting a switch from flavonols to anthocyanins. Finally, although UV-B radiation does not reach the inner fruit tissues, it down-regulated flavonoid biosynthesis in the flesh of both genotypes. This study provides, for the first time, evidence that the presence of the functional Aft allele, under UV-B radiation, redirects flavonoid synthesis towards anthocyanin production and suggests that the hp-1 allele negatively influences the response of flavonoid biosynthesis to UV-B

Effects of single or combined water deficit and aphid attack on tomato volatile organic compound (VOC) emission and plant-plant communication

Plants release a broad spectrum of volatile organic compounds (VOCs). The composition of the released VOC blend is dependent on the physiological status and, consequently, is affected by biotic and abiotic stresses. Stress-related VOCs can be perceived by different organisms, including natural enemies of herbivores and neighboring plants. Here, the responses of tomato plants (emitters) to single or combined abiotic (water stress) and biotic (aphid attack) stresses, and the effect of VOC released by emitters on neighboring unstressed plants (receivers), have been investigated. Emissions of α-pinene and methyl salicylate from plants exposed to single or combined stress, and of camphene from plants exposed to water or combined stress were significantly higher than in unstressed plants. In receivers, only the release of methyl salicylate increased when companion emitters were stressed. The expression of genes related to VOC biosynthesis and plant defense responses was unaffected or declined in water-stressed emitters, and was generally higher in receivers than in emitters. The gene coding for methyl salicylate biosynthesis was particularly active in aphid-attacked emitters and in receivers that were conditioned by the infested emitters. In addition, VOCs emitted by stressed plants induce VOC emission in unstressed receivers, and this increases attraction of parasitic wasps, which may improve protection against aphid attacks under conditions of reduced water availability

Stress-induced signaling: intraspecific plants communication mediated by volatile organic compounds (VOC)

The ph. D work is based on the investigation about the functions that volatile organic compounds (VOC) play in plant resistance to stress factors (mainly water stress and aphids) and as signal compounds in plant-plant and plant-insect interactions. A new VOC collection chamber was built to calibrate volatile organic compounds emitted from tomato plants by using the headspace-solid phase microextraction (HS-SPME) technical approach. This technique allowed to quantify for the first time dimethyl sulphonio propionate (DMSP), and, thus, dimethyl sulphide (DMS) potential emission in relation to the stress physiology of higher plants. Subsequently, HS-SPME was applied, in a different experiment, leading to the identification of C6 aldehyde (E)-2-hexenal as an induced VOC involved in water stress tolerance mechanisms of pomegranate plants. Then, an experiment was designed to investigate the signal functions of VOCs in plant-plant and tri-trophic interactions. The main result obtained is that methyl salicylate, released by stressed plants, acted as an airborne signal both attracting natural enemies of insect and inducing the up-regulation of genes controlling the defense responses in unstressed neighboring plants

Risposte di mutanti antocianici di pomodoro al trattamento in post-harvest con UV-B

Medici e nutrizionisti sottolineano sempre più frequentemente l’importanza dell’assunzione giornaliera di frutta e verdura per fornire all’organismo una serie di composti bioattivi capaci di rallentare i processi di invecchiamento cellulare che sono alla base di numerose patologie tumorali e cardiovascolari. Tuttavia, considerando che il consumo di questi alimenti è spesso inferiore a quanto raccomandato, aumentare il corredo in molecole antiossidanti di frutta e verdura rappresenta un’interessante strategia per incrementare l’apporto di composti nutraceutici con l’alimentazione. Tra i diversi approcci utilizzabili, le modificazioni delle condizioni ambientali durante la crescita della pianta e/o in post-raccolta possono rappresentare una strategia alternativa ai metodi di ingegneria genetica. In questo contesto la radiazione UV-B assume grande importanza, in quanto influisce sul sistema foto-sensoriale della pianta e gioca un ruolo fondamentale nella biosintesi e nell’accumulo di composti nutraceutici. Tra i numerosi alimenti vegetali, il pomodoro risulta essere un’ottima fonte di composti antiossidanti, grazie alla presenza di carotenoidi e composti fenolici. Obiettivo di questa tesi è stato quindi quello di verificare se trattamenti in post-raccolta con dosi controllate di radiazione UV-B siano efficaci nel promuovere l’accumulo di molecole bioprotettive ad azione antiossidante in quattro diversi genotipi di pomodoro. A tale scopo, i frutti di quattro linee di pomodoro: il mutante SA206 (Antocianina +) e il suo wild type, cv Roma, e il mutante V7112 (Antocianina -) e il relativo wild type, cv Alice, sono stati raccolti allo stadio mature green e irradiati con radiazione UV-B per un ora al giorno sino al raggiungimento dello stadio red ripe, all’interno di due celle climatiche, in condizioni controllate. L’influenza della radiazione UV-B sull’accumulo dei principali composti nutraceutici è stata valutata quantificando il contenuto in fenoli, flavonoidi e flavonoli totali mediante dosaggio spettrofotometrico e determinandone il pattern attraverso separazione in HPLC. Sempre mediante analisi in HPLC sono stati quantificati i principali carotenoidi, luteina, licopene, β-carotene e i loro precursori fitoene e fitofluene. Infine è stato determinato tramite saggio ABTS il potere antiossidante totale della frazione fenolica

Headspace-solid phase microextraction approach for dimethylsulphoniopropionate quantification in Solanum lycopersicum plants subjected to water stress

Dimethylsulphoniopropionate (DMSP) and dimethyl sulphide (DMS) are compounds found mainly in marine phytoplankton and in some halophytic plants. DMS is a globally important biogenic volatile in regulating of global sulphur cycle and planetary albedo, whereas DMSP is involved in the maintenance of plant-environment homeostasis. Plants emit minute amounts of DMS compared to marine phytoplankton and there is a need for hypersensitive analytic techniques to enable its quantification in plants. Solid Phase Micro Extraction from Head Space (HS-SPME) is a simple, rapid, solvent-free and cost-effective extraction mode, which can be easily hyphenated with GC-MS for the analysis of volatile organic compounds (VOCs). Using tomato (Solanum lycopersicum) plants subjected to water stress as a model system, we standardized a sensitive and accurate protocol for detecting and quantifying DMSP pool sizes, and potential DMS emissions, in cryoextracted leaves. The method relies on the determination of DMS free and from DMSP pools before and after the alkaline hydrolysis via HS-SPME-GC-MS. We found a significant (2.5 time) increase of DMSP content in water-stressed leaves reflecting clear stress to the photosynthetic apparatus. We hypothesize that increased DMSP, and in turn DMS, in water-stressed leaves are produced by carbon sources other than direct photosynthesis, and function to protect plants either osmotically or as antioxidants. Finally, our results suggest that SPME is a powerful and suitable technique for the detection and quantification of biogenic gases in trace amounts

Trattamenti UV-B in post-harvest inducono l’accumulo di antocianine nel mutante antocianico di pomodoro SA206-1-2-2 (aft +)

Le antocianine sono composti capaci di apportare importanti benefici alla salute umana. I frutti di pomodoro (Solanum lycopersicum L.) non producono antocianine, sebbene questa classe di flavonoidi sia sintetizzata nei tessuti vegetativi di questa specie. Al contrario, alcune specie tassonomicamente vicine, come il Solanum chilense, sono in grado di sintetizzare antocianine anche nei frutti. L‟incrocio interspecifico con Solanum chilense ha consentito il trasferimento del gene dominante AFT e l‟ottenimento di un pomodoro mutante (denominato SA206-1-2-2), capace di produrre piccole quantità di antocianine nel frutto, che si accumulano nella buccia come macchie viola concentrate in strisce verticali. L‟identità del gene AFT non è ancora stata chiarita, sebbene sia stato dimostrato che il locus aft segrega con due diversi fattori di trascrizione coinvolti nella biosintesi delle antocianine. La presente ricerca è stata condotta per capire se la produzione di antocianine da parte di questo mutante possa essere ulteriormente stimolata da parte di un trattamento con radiazione UV-B in post-harvest. E‟ stata inoltre valutata l‟influenza della radiazione UV-B sul contenuto totale di composti fenolici e flavonoidi e sui principali flavonoidi del pomodoro. Per chiarire se la risposta sia tessuto-specifica, le analisi sono state condotte separatamente in buccia e polpa dei frutti del mutante e del relativo wild type, cv Roma. I frutti, raccolti allo stadio verde maturo, sono stati esposti quotidianamente a radiazione UV-B (1 ora al giorno, 6.08 kJ m-2 d-1) fino a maturazione (stadio rosso maturo). I risultati evidenziano un effetto diverso da parte del trattamento UV-B a seconda del genotipo, del tessuto e delle molecole in esame. Infatti mentre il contenuto dei composti fenolici e dei flavonoidi subisce un incremento nella buccia della cv Roma, esso diminuisce nella polpa di entrambi i genotipi. Nel mutante SA206-1-2-2, costitutivamente più ricco di flavonoidi rispetto alla cv Roma, la radiazione UV-B ha indotto una diminuzione della concentrazione di fenoli, flavonoidi totali e rutina della buccia, ma ha portato ad un notevole aumento nei livelli delle antocianidine petunidina, delfinidina e malvidina, a suggerire uno shift metabolico verso la sintesi di antocianine in risposta alla radiazione UV-B. Sono attualmente in corso analisi molecolari volte a confermare questa ipotesi

Headspace-Solid Phase Microextraction Approach for Dimethylsulfoniopropionate Quantification in Solanum lycopersicum Plants Subjected to Water Stress

Dimethylsulfoniopropionate (DMSP) and dimethyl sulphide (DMS) are compounds found mainly in marine phytoplankton and in some halophytic plants. DMS is a globally important biogenic volatile in regulating of global sulfur cycle and planetary albedo, whereas DMSP is involved in the maintenance of plant-environment homeostasis. Plants emit minute amounts of DMS compared to marine phytoplankton and there is a need for hypersensitive analytic techniques to enable its quantification in plants. Solid Phase Micro Extraction from Head Space (HS-SPME) is a simple, rapid, solvent-free and cost-effective extraction mode, which can be easily hyphenated with GC-MS for the analysis of volatile organic compounds. Using tomato (Solanum lycopersicum) plants subjected to water stress as a model system, we standardized a sensitive and accurate protocol for detecting and quantifying DMSP pool sizes, and potential DMS emissions, in cryoextracted leaves. The method relies on the determination of DMS free and from DMSP pools before and after the alkaline hydrolysis via Headspace-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). We found a significant (2.5 tim) increase of DMSP content in water-stressed leaves reflecting clear stress to the photosynthetic apparatus. We hypothesize that increased DMSP, and in turn DMS, in water-stressed leaves are produced by carbon sources other than direct photosynthesis, and function to protect plants either osmotically or as antioxidants. Finally, our results suggest that SPME is a powerful and suitable technique for the detection and quantification of biogenic gasses in trace amounts

A biochemical and molecular dissection of fruit anthocyanins in anthocyanin-rich tomato mutant after post-harvest UV-B treatment

Phenylpropanoid consumption is well-known to be associated with a reduced risk of several human diseases. Many studies demonstrated that UV-B may influence phenylpropanoid metabolism. This work investigates how UV-B radiation can modulate the transcription of different genes involved in the phenylpropanoid pathway, in fruits of the tomato line SA206-1- 2-2 (with the anthocyanin fruit Aft gene), characterised by purple spots on fruit surface, and in its wild type, cv Roma. Fruits, harvested at mature green stage, were irradiated with UV-B (1h a day, 6.08 kJ m-2 d-1) until red ripe stage. UV-B treatment induced an overall stimulation of the flavonoid biosynthetic pathway in Roma peel. In SA206 peel, early biosynthetic genes (CHS, F3H and F3’H) were down- regulated, leading to a decrease in downstream metabolites. However, F3’5’H and DFR transcription, as well as delphinidin, petunidin and malvidin levels, increased, indicating a metabolic shift towards anthocyanin synthesis in response to UV-B irradiation

Moderate Drought Stress Induces Increased Foliar Dimethylsulphoniopropionate (DMSP) Concentration and Isoprene Emission in Two Contrasting Ecotypes of Arundo donax

The function of dimethylsulphoniopropionate (DMSP) in plants is unclear. It has been proposed as an antioxidant, osmolyte and overflow for excess energy under stress conditions. The formation of DMSP is part of the methionine (MET) pathway that is involved in plant stress responses. We used a new analytical approach to accurately quantify the changes in DMSP concentration that occurred in two ecotypes of the biomass crop Arundo donax subject to moderate drought stress under field conditions. The ecotypes of A. donax were from a hot semi-arid habitat in Morocco and a warm-humid environment in Central Italy. The Moroccan ecotype showed more pronounced reductions in photosynthesis, stomatal conductance and photochemical electron transport than the Italian ecotype. An increase in isoprene emission occurred in both ecotypes alongside enhanced foliar concentrations of DMSP, indicative of a protective function of these two metabolites in the amelioration of the deleterious effects of excess energy and oxidative stress. This is consistent with the modification of carbon within the methyl-erythritol and MET pathways responsible for increased synthesis of isoprene and DMSP under moderate drought. The results of this study indicate that DMSP is an important adaptive component of the stress response regulated via the MET pathway in A. donax. DMSP is likely a multifunctional molecule playing a number of roles in the response of A. donax to reduced water availability

Analysis of Volatile Hydrocarbons (Pentene Dimers and Terpenes) in Extra Virgin Olive Oil: Optimization by Response Surface Methodology and Validation of HS-SPME-GC-MS Method

A head space-solid phase microextraction-gas chromatography–mass spectrometery (HS-SPME-GC-MS) method for the simultaneous analysis of pentene dimers from lipoxygenase (LOX) pathway, monoterpenes, and sesquiterpenes in extra virgin olive oil (EVOO) was proposed. A Doehlert design was performed; the conditions of the HS-SPME preconcentration step (extraction temperature, extraction time, sample amount, and desorption time) were optimized by response surface methodology, allowing defining the method operable design region. A quantitative method was set up using the multiple internal standard normalization approach: four internal standards were used, and the most suitable one was selected for area normalization of each external standard. The quantitative method was successfully validated and applied to a series of monocultivar EVOOs. This is the first paper in which a quantitative method using commercial standards has been proposed for the analysis of an important class of molecules of EVOO such as pentene dimers. The optimized method is suitable for routine analysis aimed at characterizing high quality EVOOs