engleski

In situ fluorescence-based confocal imaging for detecting early stress responses of tobacco plants after exposure to silver nanoparticles or silver nitrate

Antimicrobial properties of silver and enhanced reactivity when applied in the nanoparticle form (AgNPs) led to their growing utilization in consumer products. Consequently, their increasing release into water or soil represents a potential environmental hazard and poses an indirect threat to human health. AgNP phytotoxic effects have been mainly attributed to the excess generation of reactive oxygen species (ROS), leading to the induction of oxidative stress [1]. However, most studies so far have been performed in vitro and at the whole plant or whole organ level, where oxidative stress symptoms were observed after plant exposure to stress conditions was long enough to produce extensive cell damage. In this work, detached roots of in vitro grown tobacco (Nicotiana tabacum) plants were exposed for 24 h to either AgNPs stabilized with polyvinylpyrrolidone (PVP) coating or ionic silver (AgNO3), applied in the 100 μM concentration with the aim to investigate in situ early physiological responses as first signs of stressful conditions. Generation of ROS in root cells was monitored by confocal laser scanning microscope (Leica TCS SP8) using highly sensitive and specific fluorescent probes: dihydroethidium (DHE), to detect superoxide radical (O2 –), and 2’, 7’-dichlorofluorescein-diacetate (H2DCF-DA), to detect hydrogen peroxide (H2O2) [2]. Moreover, monochlorobimane (MCB) was applied for the detection of glutathione (GSH), an important constituent of the cell antioxidant defence [3]. Additionally, propidium iodide (PI) was used as a counterstain to verify cell viability. The results showed an increased amount of H2O2 and O2 – and a decreased amount of GSH in roots exposed to both types of treatments in comparison to control, which was accompanied with an increased number of dead cells detected with PI. All observed changes were more prominent upon AgNO3- exposure compared to AgNPs. AgNPs were visualized within root cells upon both types of treatments by observing reflection and transmission with the excitation light source of 476 nm, and by employing a longer excitation wavelength (760 nm) of a multiphoton pulsed laser at which AgNP exhibit fluorescence properties. This suggests that not only AgNPs can enter roots cells, but also new AgNPs are being synthesized upon exposure to AgNO3. Fluorescence lifetime imaging microscopy (FLIM) revealed that the observed reflective and luminescent particles have a relatively short fluorescence lifetime, which could distinguish them from the natural compounds of plant cells. AgNPs were mostly found attached to the root surface and inside root hairs. Employment of transmission electron microscope (TF20, FEI Tecnai G2) coupled with energydispersive X-ray (EDX) detector confirmed AgNP localization in root cells. In conclusion, AgNPs and AgNO3 not only compromised cellular redox-homeostasis, but possibly caused cell necrosis, while different level of impact was observed depending on the form of the applied silver.

confocal microscopy, fluorescent probes, phytotoxicity, reactive oxygen species, silver nanoparticles

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