Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

Abstract

An experimental set-up which enabled non-invasive, real-time reactive oxygen species (ROS) visualization on a whole plant level was constructed. In the test organism, Lemna minor L. (common duckweed), apoplastic and symplastic oxidative stress was evaluated by exposure to menadione (50 µM), menadione (50 µM) + ascorbate (100 µM) or neither for control. Menadione (50 µM) caused a statistically significant increase in H 2 DCFDA fluorescence in the apoplast after 60 minutes of exposure. The addition of ascorbate (100 µM) in the test medium significantly decreased apoplastic oxidative stress. 50 µM menadione caused an increase in symplastic H 2 DCFDA fluorescence in 57% of fronds. The exposure of L. minor plants to both menadione and ascorbate decreased the rate of fluorescence intensity accumulation in the symplast to control levels. The method has proven to be quick and straightforward and could be applied to a range of chemicals in various physiological and toxicological plant studies. The advantages of the set-up and different possible artefacts are discussed.

Real‐time visualization of oxidative stress in a floating macrophyte Lemna minor L. exposed to cadmium, copper, menadione, and AAPH

“…We have tested a hypothesis that heavy metals (Cd and Cu) increase the production of ROS in a floating macrophyte Lemna minor . For this study we used previously devised experimental set‐up employing an ultrasensitive EMCCD camera which we used to visualize menadione‐induced oxidative stress in Lemna minor (Razinger et al, 2007b). We assessed the increase of ROS production in the apoplast by measuring the rate of fluorescence intensity increase of the test medium supplemented with a fluorescing probe dichlorofluorescein diacetate.…”

“…As previously described (Razinger et al, 2007b), this ROS visualization method's main advantages are noninvasiveness and the ability to visualize oxidative stress in whole organisms in real‐time. Additional tests with menadione and ascorbate were performed to provide further evidence of the method's usefulness.…”

“…In our previous work, we described a method employing an ultrasensitive electron multiplying charge coupled device (EMCCD) camera to visualize menadione‐induced oxidative stress (Razinger et al, 2007b). The aim of this work was to visualize heavy metal, menadione, and AAPH‐induced oxidative stress in real time on whole plants while minimizing the invasiveness of the procedure.…”