Littérature scientifique sur le sujet « Metabolomics, elicitors, secondary metabolism »

The elicitation of plant secondary metabolism may offer interesting opportunities in the framework of sustainable approaches in plant science and in terms of their ability to prime resistance to biotic and abiotic stressors. The broad metabolic reprogramming triggered by different molecular elicitors, namely salicylate (SA), polyamines (PAs), and chitosan, was comprehensively investigated using a metabolomics approach and the tomato (Solanum lycopersicum L.) as the model crop. Six different treatments were compared: a negative control (no treatments), a second negative control treated with 1 M acetic acid (the reference for chitosan, since chitosan was solubilized in acetic acid), and four molecular elicitors, 1 mM 2,1,3-benzothiadiazole (BTH, a positive control), 10 mg/mL chitosan, 0.01 mM SA, and a 0.1 mM PA (putrescine, spermidine, and spermine). All treatments determined a slight increase in biomass, in particular following PA treatment. A broad reprogramming of secondary metabolism could be observed, including membrane lipid remodeling, phenylpropanoid antioxidants, and phytohormone crosstalk. Overall, our results suggest that PAs, SA, and BTH shared a systemic acquired resistance (SAR)-related response, whereas chitosan induced a more distinct induced systemic resistance (ISR)-like jasmonate-related response. These results pave the way towards the possible use of elicitors as a sustainable tool in plant science and agriculture by increasing crop resilience to biotic and abiotic stressors without detrimental effects on plant biomass.

Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.

Metabolomics is widely applied for investigation of the correlation between metabolites and genes responsible for the synthesis of the particular sets of metabolites. In this review, we discuss metabolomics research on Hypericum perforatum (St. John’s Wort) to elucidate the overall regulation of the metabolism related to the mechanisms of natural variations and environmental stresses such as fungal infections, light stresses, and chemical elicitors. We also focus on phytochemical genomics and genomic information. St. John’s Wort is a medicinal plant with high potential of producing hypericin used for mild depression remedy, so knowledge on the biosynthetic pathway of unique metabolites is fundamental for their biotechnological commercial production. These metabolites have often complex biosynthetic pathway and it is challenging to identify all of the catalyzing enzymes. The development of metabolic systems biology could open new channels for high-speed construction and evaluation of hypotheses for cellular regulatory systems.

One of the aims of plant in vitro culture is to produce secondary plant metabolites using plant cells and organ cultures, such as cell suspensions, adventitious, and hairy roots (among others). In cases where the biosynthesis of a compound in the plant is restricted to a specific organ, unorganized systems, such as plant cell cultures, are sometimes unsuitable for biosynthesis. Then, its production is based on the establishment of organ cultures such as roots or aerial shoots. To increase the production in these biotechnological systems, elicitors have been used for years as a useful tool since they activate secondary biosynthetic pathways that control the flow of carbon to obtain different plant compounds. One important biotechnological system for the production of plant secondary metabolites or phytochemicals is root culture. Plant roots have a very active metabolism and can biosynthesize a large number of secondary compounds in an exclusive way. Some of these compounds, such as tropane alkaloids, ajmalicine, ginsenosides, etc., can also be biosynthesized in undifferentiated systems, such as cell cultures. In some cases, cell differentiation and organ formation is necessary to produce the bioactive compounds. This review analyses the biotic elicitors most frequently used in adventitious and hairy root cultures from 2010 to 2022, focusing on the plant species, the target secondary metabolite, the elicitor and its concentration, and the yield/productivity of the target compounds obtained. With this overview, it may be easier to work with elicitors in in vitro root cultures and help understand why some are more effective than others.

Il primo step del progetto di ricerca si è incentrato sulla coltivazione delle piantine di pomodoro (Solanum lycopersicum L.). le piantine sono state fatte crescere in un ambiente controllato per poter mantenere delle condizioni standard ed evitare di sottoporre a stress abiotici. Al raggiungimento della quarta foglia vera le piante sono state trattate con diversi agenti stressogeni. Come prevedeva il progetto di ricerca i trattamenti si sono incentrati su: - crescita in assenza totale di azoto; - crescita in azoto 20 Mm; - crescita in azoto 30 Mm; - trattamento con chitosano 10 gel in acido acetico; - trattamento con 1 m acido acetico (controllo negativo per il chitosano); - trattamento con fitosanitario a base di traizolici e strobilurine, quilt xcell (Syngenta) a base di azoxystrobin 141.4 g/l+ propioconazole 122.4 g/l; - trattamento con fitosanitario a base di triazolici, opinion ecna (Adama) propioconazolo 250 g/l; - trattamento con acido salicilico 0.01 mm; - trattamento con poliamine 0.1 mm; - trattamento con benzothiadiazole 137 mg/l; - trattamento con Trichoderma; - trattamento con Microrizza; - non trattato, il controllo negativo per le analisi. A seguito dei trattamenti le piante sono state sottoposte ad harvest dopo 5 giorni. Il campionamento si è basato su peso fresco e secco delle piante, raccolta di 3-5 foglie per palco. I campioni sono stati così stoccati a -18°c. Le foglie sono state poi triturate con azoto liquido di modo da mantenere intatti i metaboliti secondari presenti. Lo step successivo vede l’estrazione dei metaboliti con un protocollo di estrazione con metanolo 80%+ acido formico 1%, omogenizzazione con ultraturex, centrifugazione, filtrazione e stoccaggio a -18°C. I campioni sono stati elaborati con un untarget LC-MS/MS. L’analisi statistica dei dati raccolti è stata realizzata basandosi su HCA (hierarchical combined tree on treatment), PCA, OPLS-DA, volcano analysis e l’utilizzo combinato del database plant metabolic network, keeg pathway. Dall’analisi dei dati è emerso che, i trattamenti in presenza di un eccesso di azoto hanno portato ad una upregolazione degli alkaloids di difesa, anotcianine, flavonoidi e fitoalexine. Mentre, le piante cresciute in carenza di azoto hanno dimostrato una attivazione differenziale degli stessi pathway e maggiori condizioni di stress. Per quanto riguarda le piante di pomodoro incubate con Trichoderma hanno evidenziato una upregolazione dei pathway coinvolti nella detossificazione di composti tossici per la pianta, up regolazione anche nella produzione degli alkaloidi di difesa, della famiglia delle antocianine e fitoalexine. Una up regolazione è stata anche evidenziata nella produzione di fitosteroli. Mentre, le piante incubate con mychorrhiza non evidenziano una attivazione dei pahtway di detossificazione cellulare ma, una up regolazione nella produzione di isoflavonodi e antocianine nella forma glucosilata. Mentre, le piante trattate con controllo positivo, chitosano, una mixture di poliamine o acido sialicidico hanno mostrato come non vi sia una risposta diretta volta ad aumentarne la biomassa. Invece, ad essere aumentata in tutte le tesi è la produzione di metaboliti della classe dei pheylpropanoids ed il profilo lipidico. Tutti i cambiamenti sembrano indirizzare la pianta verso una più efficiente risposta allo stress. In ultimo, le piante sottoposte all’azione dei pesticidi hanno mostrato una diminuzione della crescita combinata con un aumento dell’attività dei processi di detossificazione ed anche in questo caso è stata riscontrata una maggiore presenza dei compositi della classe dei phenylpropanoids coinvolti in processi di difesa. In conclusione, tutti i trattamenti si sono dimostrati efficienti nell’indurre cambiamenti metabolici tali da aumentare la resistenza delle piante a condizioni avverse.
Plants are sessile organisms and therefore, they are subject to different sources of abiotic and biotic stresses. Example of abiotic stresses includes radiation, salinity, floods, drought, extremes in temperature and heavy metals. Unlike vertebrates, plants lack mobile immune cells and an adaptive immune system therefore, they have evolved different strategies to perceive and respond to the stress. Unlike vertebrates, plants lack mobile immune cells and an adaptive immune system therefore, they have evolved different strategies to perceive and respond to the stress. The first layer of plant defense systems are physical barriers, the cuticle and the cell wall, that deny access to a wide range of microbes but, also reduce water loss and protect against UV radiation. In addition to these non- specific defense mechanisms, plants have evolved a sophisticated immune response activated by the perception of highly conserved molecular features of different classes of bacterial and fungal pathogens, referred to as microbe/pathogen- associated molecular patters (M/PAMPS). This results in the activation of a defense response referred to as M/PAMPS- trigged immunity (M/PTI). Despite the activation of this line of defense, some pathogens have evolved strategies to suppress M/PTI. To overcome this infection strategy, plant have evolved specialized immune receptors encoded by resistance (r) genes (R proteins) that recognize these pathogen- specific effectors, thereby leading to an amplified secondary immune response known as effector- trigged immunity (ETI). ETI is characterized by the induction of localized programmed cell death (PCD) (referred to as the hypersensitive response or HR) in order to limit the spread of the infection, activation of defence gene expression and, induction of systemic acquired resistance (SAR) to conferring broad spectrum resistance in plants. SAR increases plant defence not only at point of infection but from whole plant. The systemic plant resistance can also be mediated by beneficial microbes living in the rhizosphere, like bacteria and fungi, this kind of plant resistance is known as induced systemic resistance (ISR). ISR is associated with enhanced ability, the so- called “priming”, to resist to stress conditions. Pricing is a mechanism that does not involve a direct activation of plant defense machinery but, it is an improved of perception and/ or amplification of defense. Priming is an adaptive, low- cost defensive measure because defense responses are only, slightly and transiently, activated by a given priming stimulus. Following the perceptions of a second stress signal (triggering stimulus), defense responses are deployed in a faster, stronger, and/or more sustained manner. Priming can involve various layers of induced defense mechanisms that are active during different levels of plant- pathogen interactions. To better understand the intracellular pathways activated upon the priming phase, molecular studies of priming strategy have been performed. These studies have recorded chromatin changes and the accumulation of mRNA of genes with a signaling role in defense, of signaling proteins and plant recognition receptors (PRRS), metabolites, and other molecular components supporting a faster, stronger, and more sustained response to a triggering stress. However, the complete elucidation of molecular pathways activated upon the perception of primed stimulus is not truly clear therefore, further studies are required. The goal of this work is to investigate on molecular mechanism of priming in the induction of ISR in plants. Metabolomics is a new field of studies that able to detect and measure all the small- molecules, metabolites, present in a given moment into a biological system. Therefore, metabolomics can be the molecular tool to detect all the changes that occur in the plant cells upon the exposure to the pricing agent and it is the perfect tool to link the metabolic change in the cell to the phenotype. To this purpose, tomato (Solanum lycopersicum L.) has been selected as model plant due to its economic interest and because of its diverse secondary metabolism. Tomato plants were grown devoid of chemical or microbiological treatments until growth stage of 9 or more leaves on main shoot unfolded and treated with different priming elicitors: Muscular mycorrhizal fungi (AMF), Trichoderma spp., benzothiadizole as positive control, triazole fungicide, a combination of strobiulurin and triazole fungicide, chitosan molecule and acetic acid (since the chitosan compound is soluble in acid medium therefore, an additional control is required), salicylic acid, polyamine mixture and in presence on nitrogen deficiency and nitrogen surplus. For plants grown under nitrogen deficiency and nitrogen surplus a different grown medium was required to avoid interference, these plants were grown in coconut coir. Tomato plants were harvested after 15 days treatments with chemical compounds and Trichoderma spp. And after 30 days for AMF inoculation. For plants grown under nitrogen deficiency/ surplus the harvest was made at growth stage of first flower bud visible. After biomass of leaves was determined together with extraction of metabolites for UHPLC/qTOF-MS analysis to investigate on molecular pathways. The study demonstrated as plants inoculated with either Arbuscular mycorrhizal fungi (AMF) or Trichoderma spp. Showed a positive effect on plant growth increasing their biomass index. The same beneficial effect on plant growth was observed in plants grown with a nitrogen surplus. While, the biomass index was not increased when plants were treated with benzothiadizole, chitosan, polyamines, salicylic acid or two pesticides, one containing only triazole and second one containing a combination of triazole and strobilurin. Notwithstanding, a broad molecular cell re-programming was also observed to include some common responses between thesis. In particular, the phenylpropanoid biosynthetic pathway was strongly elicited, with the production of defense phenolics like coumarins, bis-noryangonin, anthocyanins, and their glycosylated form in tomato under biotic stress. While, under abiotic stress (benzothiadizole, nitrogen deficiency, nitrogen surplus, chitosan, polyamines, salicylic acid, triazole compounds and a combination of triazole and strobilurin) there was an over expression of quercetin, terpenoid, amide derivate and, also anthocyanins. Another important aspect was the remodeling of membrane lipids and the production of sphingolipids as signal molecules. Under abiotic stress the sterol/phospholipid ratio increased with increasing of membrane rigidity, changes in membrane permeability and activation of stress response to abiotic factors. While, in presence of nutritional alteration (both in deficiency and surplus) the membrane composition changed decreasing the sterol to phospholipid ratio increasing in membrane fluidity probably in one case to boost nutrient uptake and in second one to avoid an intoxication due to a high amount of nitrogen in the cell. At same time, the shaping of phytohormone profiles resulted in the accumulation of auxins, cytokinins, and jasmonate under biotic stress. While, under abiotic stress there was an increasing in gibberellin and cytokinins to boost pant defenses. The treatments with pesticides lead to an increasing in brassinosteroids involved in detoxification pathways. To conclude, the establishment of symbiosis between plant and AMF and Trichoderma impacted several plant secondary metabolism processes in a fashion that supports both plant growth promotion and immunity. While the stress induced by abiotic factors were demonstrated to active similar cellular re- programming. Even if treatments do not increase plant growth, they were efficiently to increase plant survival to future stresses.

Dans le cadre de la promotion d'une viticulture durable, le développement d'alternatives écologiques à l’utilisation de pesticides de synthèse pour le contrôle des maladies de la vigne (Vitis vinifera) gagne en importance. Une des méthodes de bio-contrôle proposée est l'induction de l'immunité des plantes par l’application de substances biodégradables, non toxiques pour la santé et l'environnement, appelées éliciteurs ou stimulateurs de défense des plantes (SDP). La résistance conférée contre divers agents pathogènes peut être obtenue grâce à l’emploi de molécules utilisant le plus fréquemment les voies de signalisation de l'acide jasmonique (JA), de l'acide salicylique (SA) et/ou de l'éthylène (ET). De telles voies peuvent déclencher l'induction de gènes liés à la défense tels ceux codant des enzymes responsables de la biosynthèse des stilbènes, des métabolites phénoliques antimicrobiens parmi les plus importants des Vitacées. Au vignoble, pour contrôler les maladies, les éliciteurs peuvent être appliqués en complément des pesticides de synthèse et non en remplacement total car leur efficacité est souvent variable selon les agents pathogènes et les conditions environnementales. Afin de développer leur utilisation, des études supplémentaires qui pourraient notamment élucider leur mécanisme d'action sont nécessaires. L’objectif de cette thèse était d'étudier les réponses de la vigne à des éliciteurs de différents modes d'action, comme le jasmonate de méthyle (MeJA), impliqué dans la voie de signalisation du JA, l’acide S-méthyl ester 2,1,3-benzothiadiazole-7-carbothioïque (BTH), un analogue synthétique du SA, des phosphonates (PHOS), molécules à double action stimulateur-fongicide. Le profil des stéroïdes et des triterpénoïdes pentacycliques a été caractérisé par des analyses en chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS). Dans un premier temps, l'effet éliciteur du MeJA a été évalué dans des suspensions cellulaires (in vitro) de plusieurs cultivars de V. vinifera. Une surproduction de triterpénoïdes pentacycliques a été observée avec des différences selon la variété considérée : induction de l’accumulation de la bétuline et de l'acide oléanolique ou des phytostérols pour le Petit Verdot, le Gamay Teinturier) et le Cabernet Sauvignon, respectivement. Puis les élicitations ont été effectuées au niveau des feuilles de boutures de serre de V. vinifera cv. Cabernet Sauvignon. Un effet stimulant sur les triterpénoïdes pentacycliques liés à la défense a été démontré au détriment de la biosynthèse des stérols, composants structurels essentiels des membranes cellulaires. Par l’utilisation de puces NeoVigen et la chromatographie liquide à ultra haute performance couplée à la spectrométrie de masse (UHPLC-MS), l’induction de l’expression de gènes liés à la défense et l'accumulation de polyphénols (stilbènes, flavanols et flavonols) ont été notées suite aux trois traitements éliciteurs. La protection de la vigne conférée par les éliciteurs a été confirmée par des biotests sur disques foliaires inoculés par l’oomycète biotrophe Plasmopara viticola, l’agent responsable du mildiou. Par ailleurs, il est important d’avoir connaissance de l’impact des éliciteurs sur le métabolisme général afin d'obtenir l’effet optimal entre croissance, rendement et défense. Ainsi, une approche métabolomique utilisant la spectroscopie de résonance magnétique nucléaire du proton (RMN 1H) a été menée. Une reprogrammation similaire et/ou spécifique selon l'éliciteur considéré a été notée en particulier au niveau des glucides, des acides aminés et de certains intermédiaires du cycle de Krebs. Les recherches présentées dans cette thèse, démontrent que la compréhension approfondie de l'interaction entre l'éliciteur, les réponses moléculaires et métaboliques de la plante et le pathogène, est cruciale pour le développement de stratégies de protection efficaces basées sur l'utilisation des SDP pour contrôler les maladies de la vigne
In the frame of promoting sustainable vitiviniculture, the development of eco-friendly alternatives to synthetic chemical products for phytosanitary treatments against grapevine (Vitis vinifera) pests is gaining importance. One of the bio-control methods that can be proposed is the induction of plant immunity by using elicitors, also called plant defense stimulators (PDS), as these substances are biodegradable and, non-toxic to health and environment. A conferred resistance against various pathogens can be obtained with natural molecules acting most frequently through jasmonic acid (JA), salicylic acid (SA), and/or ethylene (ET) signaling pathways. These pathways are involved in the induction of defense-related genes such as those encoding enzymes responsible for the biosynthesis of stilbenes, which are the most important polyphenolic antimicrobial metabolites (phytoalexins) in Vitaceae. For vineyard protection, PDS can be applied as a complement for pesticides and not as a full replacement since their effectiveness is often variable according to pathogens and environmental conditions. In order to develop the strategies based on PDS use, more studies which could elucidate their mechanism of action are needed. The aim of this thesis was to examine the responses of grapevine to elicitors of different mode of action, as methyl jasmonate (MeJA), implicated in JA signaling pathway, 2,1,3-benzothiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic analogue of SA, and phosphonates (PHOS), molecules of a double stimulator-fungicide action. Due to scarce information about steroids and pentacyclic triterpenoids in grapevine, their profile after PDS treatment were characterized in different grapevine experimental models using gas chromatography-mass spectrometry (GC-MS) analyses. Firstly, the effect of elicitation with MeJA was evaluated in cell suspension cultures (in vitro) of V. vinifera. An overproduction of bioactive pentacyclic triterpenoids occurred with differences according to the cultivar studied, i.e., acumulation of betulin and oleanolic acid or phytosterols was noted in respectively Petit Verdot, Gamay Teinturier and Cabernet Sauvignon cell suspension cultures. Then, elicitations were effectuated on the leaves of V. vinifera cv. Cabernet Sauvignon greenhouse cuttings. A stimulatory effect on the potentially defense-related pentacyclic triterpenoids at the expense of the biosynthesis of sterols, which are essential structural components of cell membranes, was shown. By the use of NeoVigen microarrays, and ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS), the accumulation of defense-related transcripts and polyphenols (stilbenes, flavanols and flavonols) were noted after the three elicitors treatments. Grapevine protection conferred by these elicitors was confirmed on foliar discs against the biotrophic oomycete Plasmopara viticola, the causal agent of downy mildew. Furthermore, the impact of PDS on primary metabolism should be evaluated in order to ensure, in the longer term, the best trade-off between growth, yield and defense. Thus, a thorough metabolomic approach using proton nuclear magnetic resonance spectroscopy (1H-NMR) was performed. A reprogramming similar and/or specific to the elicitor applied was noted, particularly within carbohydrates, amino acids, and some of the Krebs cycle intermediates. The research presented in the current dissertation revealed that the thorough comprehension of the interaction between elicitor, plant molecular and metabolic responses and pathogen, is crucial for the development of effective protection strategies based on the use of PDS for grapevine diseases control

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Les Streptomyces sont des bactéries filamenteuses à Gram positif que l’on retrouve dans les couches superficielles du sol. La souche modèle S. coelicolor M145 est capable de produire des antibiotiques et accumuler de faibles quantités de triacylglycérol lorsqu’elle est mise en culture avec le glucose ou le glycérol comme source de carbone. La souche produit de l’actinorhodine seulement en culture avec le glycérol comme source de carbone, et nous assistons à une accumulation d’acides gras, notamment sous forme d’esters d’acide oléique avec l’une ou l’autre source de carbone. Ces accumulations d’acides gras ne durent que pendant une certaine période de temps, à l’issu de laquelle l’accumulation s’arrête, et on assiste à la production de métabolites secondaires qui peut s’accompagner de la consommation des acides gras précédemment accumulés. Ces observations suggèrent d’une part que la source de carbone est un effecteur différentiel dans la production de métabolites secondaire, et que d’autre part le métabolisme des lipides est lié au métabolisme secondaire. Ce travail de thèse va tenter d’établir les liens entre le métabolisme primaire et le métabolisme secondaire chez S.coelicolor via le métabolisme des lipides. Pour se faire, nous avons dans un premier temps défini un milieu synthètique, ainsi que des stratégies d’alimentation de type fed-batch en bioréacteur, pour permettre d’obtenir d’une part une phase de croissance exponentielle, et d’autre part une phase de croissance non exponentielle et imposée par une limitation en azote. Cette limitation nutritionnelle est classiquement utilisée à la fois lors des études de l’accumulation de lipides chez les microorganismes oléagineux tels que Yarrowia lipilytica ou Rhodotorula glutinis, et aussi dans le cas d’études de la production d’antibiotiques par les Streptomyces. L’étude du métabolome couplée à l’étude du protéome nous a permis d’établir la direction générale des flux globaux de carbone, d’énergie et de pouvoir réducteur lors des différentes phases de croissances. Ces travaux démontrent une hétérogénéité métabolique dans la population bactérienne totale, avec la présence de 2 à 3 sous-populations différentes pouvant coexister. Les évolutions temporelles du protéome et notamment des régulateurs transcritptomiques montrent des réactions tardives, très certainement dues à la présence d’effecteurs intracellulaires accumulés, ainsi qu’au dynamisme de la culture
Streptomyces are filamentous Gram positive bacteria found in the soil upper layers. The model strain S. coelicolor M145 can produce antiobiotics and accumulate low levels of triacylglycerol when cultivated with glucose or glycerol as carbon source. This strain produces actinorhodin only when glycerol is used as carbone source, but the fatty acids accumulation as esterified oleic acid occurs with both carbon sources. However, the fatty acids accumulation only last for a short period of time, and afterward, a production of secondary metabolites is observed and the previously accumulated fatty acids are consummed. Those results suggest that the carbon source act as an effector for the production of secondary metabolites and the lipids metabolism is some ways linked to the secondary metabolism. With this work, we will try to establish the links between the primary and secondary metabolism via the lipids metabolism with S. coelicolor. First, in order to obtain cultures with an exponential growth phase and a non-exponential growth phase under a nitrogen limitation, a synthetic media and fed-batch feeding strategies have been designed. The nitrogen limitation is usually used to study lipids accumulation with oleaginous microorganisms such as Yarrowia lipilytica or Rhodotorula glutinis, and also during studies of antibiotics production with Streptomyces. The metabolomics study paired with the proteomics study made possible to establish the global directions of carbon, energy and reduced power fluxes during all the obtained growth phases. This work also showed that the microbial population is heterogeneous and 2 to 3 subpopulations can coexist with both carbon sources. The proteomics temporal changes and in particular of transciptional regulators show some late reactions to the nitrogen limitation especially when glycerol is used, probably because of the intracellular accumulation of effector compounds over time, and because of growth kinetics

In the frame of promoting sustainable vitiviniculture, the development of eco-friendly alternatives to synthetic chemical products for phytosanitary treatments against grapevine (Vitis vinifera) pests is gaining importance. One of the bio-control methods that can be proposed is the induction of plant immunity by using elicitors, also called plant defense stimulators (PDS), as these substances are biodegradable and, non-toxic to health and environment. A conferred resistance against various pathogens can be obtained with natural molecules acting most frequently through jasmonic acid (JA), salicylic acid (SA), and/or ethylene (ET) signaling pathways. These pathways are involved in the induction of defense-related genes such as those encoding enzymes responsible for the biosynthesis of stilbenes, which are the most important polyphenolic antimicrobial metabolites (phytoalexins) in Vitaceae. For vineyard protection, PDS can be applied as a complement for pesticides and not as a full replacement since their effectiveness is often variable according to pathogens and environmental conditions. In order to develop the strategies based on PDS use, more studies which could elucidate their mechanism of action are needed. The aim of this thesis was to examine the responses of grapevine to elicitors of different mode of action, as methyl jasmonate (MeJA), implicated in JA signaling pathway, 2,1,3-benzothiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic analogue of SA, and phosphonates (PHOS), molecules of a double stimulator-fungicide action. Due to scarce information about steroids and pentacyclic triterpenoids in grapevine, their profile after PDS treatment were characterized in different grapevine experimental models using gas chromatography-mass spectrometry (GC-MS) analyses. Firstly, the effect of elicitation with MeJA was evaluated in cell suspension cultures (in vitro) of V. vinifera. An overproduction of bioactive pentacyclic triterpenoids occurred with differences according to the cultivar studied, i.e., acumulation of betulin and oleanolic acid or phytosterols was noted in respectively Petit Verdot, Gamay Teinturier and Cabernet Sauvignon cell suspension cultures. Then, elicitations were effectuated on the leaves of V. vinifera cv. Cabernet Sauvignon greenhouse cuttings. A stimulatory effect on the potentially defense-related pentacyclic triterpenoids at the expense of the biosynthesis of sterols, which are essential structural components of cell membranes, was shown. By the use of NeoVigen microarrays, and ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS), the accumulation of defense-related transcripts and polyphenols (stilbenes, flavanols and flavonols) were noted after the three elicitors treatments. Grapevine protection conferred by these elicitors was confirmed on foliar discs against the biotrophic oomycete Plasmopara viticola, the causal agent of downy mildew. Furthermore, the impact of PDS on primary metabolism should be evaluated in order to ensure, in the longer term, the best trade-off between growth, yield and defense. Thus, a thorough metabolomic approach using proton nuclear magnetic resonance spectroscopy (1H-NMR) was performed. A reprogramming similar and/or specific to the elicitor applied was noted, particularly within carbohydrates, amino acids, and some of the Krebs cycle intermediates. The research presented in the current dissertation revealed that the thorough comprehension of the interaction between elicitor, plant molecular and metabolic responses and pathogen, is crucial for the development of effective protection strategies based on the use of PDS for grapevine diseases control.
Rozwój przyjaznych dla środowiska alternatywnych metod ochrony winorośli (Vitis vinifera) przeciw szkodnikom zyskuje na znaczeniu w kontekście zapobiegania nadmiernemu użyciu pestycydów i promowania zrównoważonego rolnictwa. Indukcja naturalnej obrony roślin poprzez działanie elicytorów (stymulatorów odporności roślin) jest jedną z najbardziej obiecujących metod kontroli biologicznej, jako że opiera się na zastosowaniu substancji biodegradowalnych i nietoksycznych dla środowiska i zdrowia ludzi. Elicytory zaaplikowane na roślinę wyzwalają w niej mechanizmy obronne, prowadząc do rozwijania odporności na kolejne ataki patogenów. Percepcja elicytora uruchamia szlaki sygnałowe, z których najbardziej kluczowe są te związane z kwasem salicylowym (SA), kwasem jasmonowym (JA) lub etylenem (ET). Konsekwencje aktywowania kaskady reakcji odpornościowych to m.in. indukcja ekspresji genów związanych z obroną, np. kodujących białka związane z patogenezą (PR), czy enzymy odpowiedzialne za biosyntezę wyspecjalizowanych metabolitów o właściwościach przeciwdrobnoustrojowych (przede wszystkim stilbenów u roślin z rodziny Vitaceae). Stymulatory odporności roślin są obecnie stosowane jako uzupełnienie pestycydów, a nie jako środek zastępczy, ponieważ ich skuteczność jest często zmienna w zależności od konkretnego patogena i warunków środowiskowych. W celu opracowania strategii ochrony winorośli przed chorobami całkowicie opartych na elicytorach, potrzeba jest wielu badań, które pozwolą m.in. wyjaśnić mechanizm działania tych środków. Celem niniejszej pracy było zbadanie odpowiedzi winorośli na elicytory o różnej aktywności biologicznej: jasmonian metylu (MeJA), związany ze szlakiem sygnałowym JA, benzotiadiazol (BTH), syntetyczny analog SA oraz sole fosforanowe (PHOS), o działaniu stymulującym, jak i grzybobójczym. Profil steroidów i triterpenoidów pentacyklicznych winorośli scharakteryzowano za pomocą chromatografii gazowej sprzężonej ze spektrometrią mas (GC-MS). Potencjalny wpływ MeJA na profil triterpenoidów oceniono w hodowlach zawiesin komórkowych in vitro V. vinifera. W zależności od badanej odmiany, zaobserwowano wzmożoną biosyntezę triterpenoidów pentacycklicznych, takich jak betulina (Petit Verdot) i kwas oleanolowy (Gamay Teinturier), a także fitosteroli (Cabernet Sauvignon). W elicytowanych liściach szklarniowych sadzonek Cabernet Sauvignon, wykazano konkurencyjność szlaków biosyntezy triterpenoidow, tzn. zwiększona akumulacja triterpenoidow pentacyklicznych, związanych z chemiczną obroną roślin, odbyła się kosztem biosyntezy steroli, związków niezbędnych dla budowy i funkcjonowania błon komórkowych. Dzięki analizom z wykorzystaniem mikromacierzy NeoVigen i ultrasprawnej chromatografii cieczowej ze spektrometrią mas (UHPLC-MS), w liściach poddanych elicytacji stwierdzono akumulację transkryptów związanych z obroną oraz zwiększoną zawartość polifenoli (stilbenów, flawanoli i flawonoli). Biotesty na krążkach liściowych wykazały, że badane elicytory nadały odporność liściom przeciwko Plasmopara viticola, tj. patogenowi wywołującemu mączniaka rzekomego. Ponadto, badanie wpływu elicytorów na metabolizm pierwotny jest istotny w celu zapewnienia roślinie swoistego kompromisu między aktywowaniem kosztownych energetycznie mechanizmów obronnych, a utrzymaniem prawidłowego funkcjonowania podstawowych procesów fizjologicznych. Badania metabolomiczne z wykorzystaniem spektroskopii protonowego jądrowego rezonansu magnetycznego (1H-NMR) wykazały szereg istotnych zmian w profilu węglowodanów, aminokwasów i niektórych substratów cyklu Krebsa w liściach poddanych elicytacji. Wyniki badań przedstawione w niniejszej rozprawie wykazują, iż dokładne zrozumienie interakcji między elicytorem, odpowiedzią molekularną i metaboliczną rośliny a patogenem, ma kluczowe znaczenie dla rozwoju skutecznych strategii obrony winorośli przed chorobami, opartych na stymulatorach odporności roślin.

AbstractPlants encounter various nanomaterials (NMs) as pesticides and fertilizers. It is also possible that nanomaterials reach plants as waste from consumer products and industry. The effects of such NMs on plants have been widely studied, and both positive and negative effects of NMs on plant growth and development have been reported. Recent metabolomics studies suggest that nanoparticles affect the concentration of secondary metabolites in plants by modulating reactive nitrogen/oxygen species, gene expression, and signaling pathways. Secondary metabolites are plant compounds that accumulate in plants through their secondary metabolism. To date, more than 200,000 defined structures of secondary metabolites have been identified, among which many of them possess antibacterial, antifungal, antiviral, anti-inflammatory, hepatoprotective, antidepressant, antioxidant, neuroprotective, and anticancer properties. The application of elicitors is a simple strategy to increase the production of secondary metabolites in plant cell and tissues. The ability of nanomaterials to induce plant secondary metabolism has recently been exploited in the elicitation of pharmaceutically important compounds from various plant species. The ability of different NMs to induce the accumulation of different classes of compounds in the same plant species has also been accomplished. The molecular mechanisms behind the effects of NMs on plant secondary metabolism revealed the putative genes involved in NM-mediated elicitation of various plant compounds in several reports. This chapter reviews the current understanding of the effects of nanoparticles on plant secondary metabolism and the elicitation of pharmacologically important compounds from plant species.

Interferon-gamma belongs to a large family of cytokines – multifunctional secreted proteins involved in animal non-specific immune response. Previously inbred lines of Nicotiana tabacum L. plants harboring a heterologous gene of bovine interferon-gamma Bt-sIFNG under the control of a constitutive 35S CaMV promoter have been created by Agrobacterium-mediated genetic transformation. The antiviral and immunomodulatory activities of plant-produced interferon-gamma in bovine cell culture and laboratory animals (mice) were observed. A state-of-the-art GS-MS technique has been used to identify the possible effect of the transformation on the plant’s metabolome. Total profiles included 350 metabolites from leaves, among which 150 substances were identified up to their class and 80 up to the exact metabolite. Metabolite profiling revealed that plants able to synthesize interferon-gamma are characterized by a higher level of amino acids and other substances involved in nitrogen metabolism. In transgenic plants intensification of the secondary metabolism was also detected. Some alterations were distinguished in plant metabolome depending on cultivation conditions.

Metabolomics is one of the new field of “Omics” approach and the youngest triad of system biology, which provides a broad prospective of how metabolic networks are controlled and indeed emerged as a complementary tool to functional genomics with well-established technologies for genomics, transcriptomics and proteomics. Though, metabolite profiling has been carried out for decades, owing to decisive mechanism of a molecule regulation, the importance of some metabolites in human regimen and their use as diagnostic markers is now being recognized. Plant metabolomics therefore aims to highlight the characterization of metabolite pool of a plant tissue in response to its environment. Seagrassses, a paraphyletic group of marine hydrophilous angiosperms which evolved three to four times from land plants back to the sea. Seagrasses share a number of analogous acquired metabolic adaptations owing to their convergent evolution, but their secondary metabolism varied among the four families that can be considered as true seagrasses. From a chemotaxonomic point of view, numerous specialized metabolites have often been studied in seagrasses. Hence, this chapter focus the metabolome of seagrasses in order to explore their bioactive properties and the recent advancements adopted in analytical technology platforms to study the non-targeted metabolomics of seagrasses using OMICS approach.

The goals of the present research proposal were to elucidate the physiological and molecular basis of the regulation of stilbene metabolism in grape, against the background of (i) grape metabolic network behavior in response to drought and of (ii) varietal diversity. The specific objectives included the study of the physiology of the response of different grape cultivars to continuous WD; the characterization of the differences and commonalities of gene network topology associated with WD in berry skin across varieties; the study of the metabolic response of developing berries to continuous WD with specific attention to the stilbene compounds; the integration analysis of the omics data generated; the study of isolated drought-associated stress factors on the regulation of stilbene biosynthesis in plantaand in vitro. Background to the topic Grape quality has a complex relationship with water input. Regulated water deficit (WD) is known to improve wine grapes by reducing the vine growth (without affecting fruit yield) and boosting sugar content (Keller et al. 2008). On the other hand, irregular rainfall during the summer can lead to drought-associated damage of fruit developmental process and alter fruit metabolism (Downey et al., 2006; Tarara et al., 2008; Chalmers et al., 792). In areas undergoing desertification, WD is associated with high temperatures. This WD/high temperature synergism can limit the areas of grape cultivation and can damage yields and fruit quality. Grapes and wine are the major source of stilbenes in human nutrition, and multiple stilbene-derived compounds, including isomers, polymers and glycosylated forms, have also been characterized in grapes (Jeandet et al., 2002; Halls and Yu, 2008). Heterologous expression of stilbenesynthase (STS) in a variety of plants has led to an enhanced resistance to pathogens, but in others the association has not been proven (Kobayashi et al., 2000; Soleas et al., 1995). Tomato transgenic plants harboring a grape STS had increased levels of resveratrol, ascorbate, and glutathione at the expense of the anthocyanin pathways (Giovinazzo et al. 2005), further emphasizing the intermingled relation among secondary metabolic pathways. Stilbenes are are induced in green and fleshy parts of the berries by biotic and abiotic elicitors (Chong et al., 2009). As is the case for other classes of secondary metabolites, the biosynthesis of stilbenes is not very well understood, but it is known to be under tight spatial and temporal control, which limits the availability of these compounds from plant sources. Only very few studies have attempted to analyze the effects of different environmental components on stilbene accumulation (Jeandet et al., 1995; Martinez-Ortega et al., 2000). Targeted analyses have generally shown higher levels of resveratrol in the grape skin (induced), in seeded varieties, in varieties of wine grapes, and in dark-skinned varieties (Gatto et al., 2008; summarized by Bavaresco et al., 2009). Yet, the effect of the grape variety and the rootstock on stilbene metabolism has not yet been thoroughly investigated (Bavaresco et al., 2009). The study identified a link between vine hydraulic behavior and physiology of stress with the leaf metabolism, which the PIs believe can eventually lead to the modifications identified in the developing berries that interested the polyphenol metabolism and its regulation during development and under stress. Implications are discussed below.