Real-time electrochemical detection of extracellular nitric oxide in tobacco cells exposed to cryptogein, an elicitor of defence responses

It was previously reported that cryptogein, an elicitor of defence responses, induces an intracellular production of nitric oxide (NO) in tobacco. Here, the possibility was explored that cryptogein might also trigger an increase of NO extracellular content through two distinct approaches, an indirect method using the NO probe 4,5-diaminofluorescein (DAF-2) and an electrochemical method involving a chemically modified microelectrode probing free NO in biological media. While the chemical nature of DAF-2-reactive compound(s) is still uncertain, the electrochemical modified microelectrodes provide real-time evidence that cryptogein induces an increase of extracellular NO. Direct measurement of free extracellular NO might offer important new insights into its role in plants challenged by biotic stresses

The Pseudomonas fluorescens siderophore pyoverdine weakens arabidopsis thaliana defense in favor of growth in iron-deficient conditions

Pyoverdines are siderophores synthesized by fluorescent Pseudomonas spp. Under iron-limiting conditions, these high-affinity ferric iron chelators are excreted by bacteria in the soil to acquire iron. Pyoverdines produced by beneficial Pseudomonas spp. ameliorate plant growth. Here, we investigate the physiological incidence and mode of action of pyoverdine from Pseudomonas fluorescens C7R12 on Arabidopsis (Arabidopsis thaliana) plants grown under iron-sufficient or iron-deficient conditions. Pyoverdine was provided to the medium in its iron-free structure (apo-pyoverdine), thus mimicking a situation in which it is produced by bacteria. Remarkably, apo-pyoverdine abolished the iron-deficiency phenotype and restored the growth of plants maintained in the iron-deprived medium. In contrast to a P. fluorescens C7R12 strain impaired in apo-pyoverdine production, the wild-type C7R12 reduced the accumulation of anthocyanins in plants grown in iron-deficient conditions. Under this condition, apo-pyoverdine modulated the expression of around 2,000 genes. Notably, apo-pyoverdine positively regulated the expression of genes related to development and iron acquisition/redistribution while it repressed the expression of defense-related genes. Accordingly, the growth-promoting effect of apo-pyoverdine in plants grown under iron-deficient conditions was impaired in iron-regulated transporter1 and ferric chelate reductase2 knockout mutants and was prioritized over immunity, as highlighted by an increased susceptibility to Botrytis cinerea This process was accompanied by an overexpression of the transcription factor HBI1, a key node for the cross talk between growth and immunity. This study reveals an unprecedented mode of action of pyoverdine in Arabidopsis and demonstrates that its incidence on physiological traits depends on the plant iron status

A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses

Présentation du métier de chercheur dans le cadre de "Les filles et la Science"

National audienc

Présentation du métier de chercheur dans le cadre de "Les filles et la Science"

National audienc

Les femmes, les sciences et l’Europe. Intervention à la conférence-débat

National audienc

Présentation du métier de chercheur dans le cadre de "Les filles et la Science"

International audienc

Etude du rôle du monoxyde d'azote (NO) dans la réponse d'Arabidopsis thaliana au cadmium (liens avec l'homéostasie du fer)

Ce travail est une contribution à l'étude du mode d'action du monoxyde d'azote (NO) dans la réponse des plantes aux stress (a)biotiques. Nous avons analysé le rôle du NO dans la toxicité du cadmium (Cd) chez Arabidopsis thaliana. Les plantes ayant absorbé du Cd par les racines produisent du NO dans les racines et les feuilles. Le NO participe à l'inhibition de la croissance des racines induite par le Cd, favorise son absorption et contribue à la réduction de la concentration de calcium (Ca). La production de NO serait la conséquence de la réduction de la concentration en fer. Le NO agirait ensuite comme un signal de carence en fer et activerait l'expression de gènes associés au transport du fer, facilitant ainsi l import du Cd. Le NO pourrait aussi promouvoir l activation de canaux/transporteurs de Ca perméables au Cd et/ou favoriser une compétition entre Cd et Ca. En conclusion, le NO est un médiateur de la toxicité du Cd. Parmi les gènes cibles du NO figure AtNAS4 codant une enzyme catalysant potentiellement la synthèse de nicotianamine (NA). Nous avons caractérisé un mutant d insertion de ce gène qui arbore un taux de NA réduit, indiquant qu AtNAS4 est une source de NA dans les feuilles. L étude du mutant a révélé qu AtNAS4 joue un rôle clé dans l homéostasie du fer, dans la résistance au Cd et en réponse à l infection par Erwinia chrysanthemi. Enfin, nous avons développé une méthode électrochimique de dosage du NO dans le milieu extracellulaire de suspensions cellulaires de tabac. Cette méthode quantitative qui s est avérée sélective et sensible a permis de confirmer la capacité de la cryptogéine, un éliciteur des réactions de défense, à déclencher une production de NO.Here, we investigated the role of nitric oxide (NO) in plant responses to (a)biotic stresses. In a first part, we analyzed the synthesis and role of NO in plants exposed to cadmium (Cd), a non essential and toxic metal. We demonstrated that Cd induces NO synthesis in roots and leaves of Arabidopsis thaliana treated at the root level. NO contributes to Cd-triggered inhibition of root growth and to Cd uptake and is also responsible for Cd-induced inhibition of root Ca accumulation. Taken together, our results suggest that NO contributes to Cd toxicity by favouring Cd versus Ca uptake and by initiating a cellular pathway resembling those activated upon iron deprivation via the activation of expression of genes related to iron uptake. One of the NO-modulated genes in response to Cd treatment is NAS4 (nicotianamine synthase 4) encoding an enzyme potentially catalysing the formation of nicotianamine (NA), a metal chelator. We showed that atnas4, a mutant carrying a T-DNA insertion in AtNAS4, displays a reduced NA content in leaves, indicating that AtNAS4 is a major enzymatic source for NA. This mutant exhibits a hypersensitivity to iron deficiency, to Cd treatment and to infection by the bacterial pathogen Erwinia chrysanthemi. We conclude that AtNAS4 plays a central role in iron homeostasis as well as in Cd and E. chrysanthemi resistance in A. thaliana. The second part of this study dealt with an electrochemical method we developed in order to probe free NO in the extracellular medium of tobacco suspension cells in response to cryptogein, an elicitor of defense responses. This technique provided real-time evidence that cryptogein is able to induce an increase of extracellular NO.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

NO contributes to cadmium toxicity in Arabidopsis thaliana by mediating an iron deprivation response

Several studies have revealed that nitric oxide (NO), an endogenous mediator in diverse physiological processes, is produced in plants exposed to the toxic metal cadmium (Cd). It was first shown that exogenously applied NO protects plant tissues against the oxidative damages triggered by Cd, suggesting a putative role for NO in counteracting the deleterious effects of Cd. More recently, our team as well as other laboratories challenged this view and demonstrated that endogenously produced NO promotes the metal-induced reduction of root growth. We investigated more thoroughly the role of NO in mediating Cd effects in roots. We have shown that in Arabidopsis thaliana, the Cd-mediated NO production is sensitive to mammalian NO synthase inhibitors and occurs downstream of IRT1, a major iron transporter also involved in the uptake of Cd. Our data support a model in which this production might be related to the iron deprivation caused by Cd. Accordingly, we found that NO upregulates the expression of genes encoding proteins related to iron acquisition, including IRT1. This process might explain the ability of NO to amplify Cd uptake and, consequently, the toxic effects of the metal