Mitochondrial behaviour in the early stages of ROS stress leading to cell death in Arabidopsis thaliana

Abstract

Background and aims: Reactive oxygen species (ROS) are involved in triggering cell death. To visualize mitochondrial behaviour under ROS stress, transgenic arabidopsis plants possessing mitochondrial-targeted GFP (S65T) were studied.

Methods: Arabidopsis leaves were treated with ROS and ROS-inducing chemicals such as hydrogen peroxide, paraquat and menadione. Microscopic observations were carried out using a confocal laser scanning microscope system, and electrolyte leakage was also monitored.

Key results: After treatment, mitochondria showed morphological changes from a bacillus-like to a round shape. The size of mitochondria treated with H(2)O(2) decreased by half compared with controls. Concurrently, cytoplasmic streaming was blocked and mitochondria eventually swelled. Treatment of leaves with butanedione monoxime, an inhibitor of myosin ATPase, resulted in similar behaviour of mitochondria to that under ROS stress.

Conclusions: The results indicate that morphological changes of mitochondria and cessation of cytoplasmic streaming may interact, and this phenomenon is one of the features of ROS stress-induced cell death.

Fig. 1.

Evaluation of ion leakage in arabidopsis leaves treated with different ROS-inducing chemicals. (A) Effects of concentrations of ROS-inducing chemicals on ion leakage. Electrolyte leakage from leaf discs prepared from 3-week-old seedlings was measured after 24 h in H₂O₂ (0–150 mm), paraquat (0–0·6 µm) or menadione (0–100 µm). Data are means ± s.d. of three experiments. (B) Time course analysis of electrolyte leakage from leaf discs. Three leaf discs prepared from 3-week-old plants were treated with 100 mm H₂O₂, 0·3 µm paraquat or 60 µm menadione for 72 h. Controls were treated with water. Reported values are average ± s.d. of three or more experiments.

Fig. 2.

Mitochondrial changes under ROS stress. (A) Morphology of mitochondria incubated in ROS-inducing chemicals for 24 and 72 h. Epidermal cells of leaf discs obtained from transgenic mt-GFP arabidopsis were examined using a confocal laser scanning microscope. Leaf discs were treated with 100 mm H₂O₂, 0·3 µm paraquat or 60 µm menadione solutions and incubated at 23 °C under continuous light. Controls were treated with water. Scale bar = 10 µm. (B) Comparison of mitochondrial area and maximum diameter. The size and the area were quantified using Image-Pro® Plus version 4·0 (Media Cybernetics) based on the images of cells treated with 100 mm H₂O₂ (1 d).

Fig. 3.

Movement of mitochondria in epidermal cells of arabidopsis leaves expressing mt-GFP, which had been treated with ROS-inducing chemicals H₂O₂ (100 mm) or paraquat (0·3 µm) for 24 h or menadione (60 µm) for 72 h. Samples were observed using a confocal laser scanning microscope at 488 nm excitation wavelength to detect mt-GFP. Images were taken at 5-s intervals. Scale bars = 10 µm. Supplementary movie reproduces images of Fig. 3. The speed is 50-fold faster than actual.

Fig. 4.

Effects of a myosin ATPase inhibitor on arabidopsis mitochondria. (A) Leaf discs 3 d after BDM treatment. Three leaf discs were submerged in BDM solution (0–20 mm) and incubated at 23 °C under continuous light. (B) Morphological changes of mitochondria in epidermal cells of arabidopsis leaves treated with BDM. Leaf discs obtained from 1-month-old mt-GFP plants were treated with 20 mm BDM for 1 h prior to observation by confocal laser scanning microscopy. Upper panels indicate controls without BDM treatment. Images were taken at 5-s intervals. Scale bar = 5 µm.