Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants

Ledoux, Quentin; Veys, Pascal; Cutsem, Pierre Van; Mauro, Sergio; Lucaccioni, Fabio; Markó, István E.

doi:10.4161/psb.26827

Cited by 9 publications

(6 citation statements)

References 23 publications

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“…They have been widely used for live-cell imaging in non-cell wall systems 18 . Ledoux et al (2013) 19 describe the application of this probe in Arabidospsis thaliana . However, enhancement of fluorescence upon oxidation of boronate-based dyes is irreversible and, therefore, limits its applicability for analysis of H 2 O 2 dynamics.…”

Section: Discussionmentioning

confidence: 99%

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum

Mentges

Bormann

2015

Sci Rep

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Balanced dynamics of reactive oxygen species in the phytopathogenic fungus Fusarium graminearum play key roles for development and infection. To monitor those dynamics, ratiometric analysis using the novel hydrogen peroxide (H2O2) sensitive fluorescent indicator protein HyPer-2 was established for the first time in phytopathogenic fungi. H2O2 changes the excitation spectrum of HyPer-2 with an excitation maximum at 405 nm for the reduced and 488 nm for the oxidized state, facilitating ratiometric readouts with maximum emission at 516 nm. HyPer-2 analyses were performed using a microtiter fluorometer and confocal laser scanning microscopy (CLSM). Addition of external H2O2 to mycelia caused a steep and transient increase in fluorescence excited at 488 nm. This can be reversed by the addition of the reducing agent dithiothreitol. HyPer-2 in F. graminearum is highly sensitive and specific to H2O2 even in tiny amounts. Hyperosmotic treatment elicited a transient internal H2O2 burst. Hence, HyPer-2 is suitable to monitor the intracellular redox balance. Using CLSM, developmental processes like nuclear division, tip growth, septation, and infection structure development were analyzed. The latter two processes imply marked accumulations of intracellular H2O2. Taken together, HyPer-2 is a valuable and reliable tool for the analysis of environmental conditions, cellular development, and pathogenicity.

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Section: Discussionmentioning

confidence: 99%

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum

Mentges

Bormann

2015

Sci Rep

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“…The generation of ROS was monitored according to the methods of Ledoux et al and Santos et al [31, 32], using 2′, 7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) as a probe. Samples of suspension cell cultures (2 ml), at day 0, were incubated in 24-well cell culture cluster with 5 μM H 2 DCFDA.…”

Section: Methodsmentioning

confidence: 99%

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures

et al. 2016

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BackgroundSenescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light.Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days.ResultsDarkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays.ConclusionsEndogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0917-y) contains supplementary material, which is available to authorized users.

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“…Recently, a new family of o-hydroxyamino-triarylpyrylium salts-based probes for NO detection was reported (Beltrán et al, 2014 ). A new fluorescent probe ContPY1 was prepared for investigations of hydrogen peroxide and tested in Arabidopsis , both on cultured cells and on leaves (Ledoux et al, 2013 ). Also, a single fluorescent probe, capable of simultaneous monitoring of both NO and H 2 O 2 endogenously produced in living macrophages (Yuan et al, 2012 ) was synthesized.…”

Section: Dawn Of Reliable Ros and Rns Imaging?mentioning

confidence: 99%

Re-Evaluation of Imaging Methods of Reactive Oxygen and Nitrogen Species in Plants and Fungi: Influence of Cell Wall Composition

Sedlářová

Luhová

2017

Front. Physiol.

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Developmental transitions and stress reactions in both eukaryotes and prokaryotes are tightly linked with fast and localized modifications in concentrations of reactive oxygen and nitrogen species (ROS and RNS). Fluorescent microscopic analyses are widely applied to detect localized production of ROS and RNS in vivo. In this mini-review we discuss the biological characteristics of studied material (cell wall, extracellular matrix, and tissue complexity) and its handling (concentration of probes, effect of pressure, and higher temperature) which influence results of histochemical staining with “classical” fluorochromes. Future perspectives of ROS and RNS imaging with newly designed probes are briefly outlined.

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Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants

Cited by 9 publications

References 23 publications

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures

Re-Evaluation of Imaging Methods of Reactive Oxygen and Nitrogen Species in Plants and Fungi: Influence of Cell Wall Composition

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