The level and redox status of glutathione is a good indicator for the rate of oxidative stress and eco-toxicological injury in plant cells and subcellular organelles. Thus the determination of GSH and its redox status has special importance. A variety of spectrophotometric and HPLC methods are available to measure glutathione (GSH). The spectrophotometric DTNB-GSH recycling assay is specific due to the application of glutathione reductase, it is rather quick and easy to perform, not surprising that it is rather popular. In the present study we make an attempt to compare the DTNB-GSH recycling assay and the more sophisticated, but difficult monochlorobimane (mBCl)-HPLC method to choose the one that best suits for eco-toxicological and plant stress investigations. We found that the acidification by sulphosalicylic acid (SSA) used for the stabilization of samples for DTNB-GSH recycling assay gives lower efficiency to this method than the formation of mBCl-GSH fluorescent conjugate. The measurable GSH contents were lower in the case of DTNB-GSH recycling assay than in the case of GSH-mBCl conjugates determined by HPLC with fluorescence detection. The auto-oxidation could almost perfectly be prevented by the presence of mBCl in the organelle isolation buffer. Furthermore, this way the reduced GSH content of organelles could be determined much more precisely. However, it is worth to note that the application of mBCl significantly elevates the cost of GSH determination, especially in case of cell organelles.

The iron dependent, programmed cell death, ferroptosis was described first in tumour cells. It showed distinct features from the already known cell death forms such as apoptosis, necrosis and autophagy. The caspase independent cell death could be induced by the depletion of glutathione by erastin or by the inhibition of the lipid peroxide scavenger enzyme GPX4 by RSL3 and it was accompanied by the generation of lipid reactive oxygen species. Recently, ferroptosis-like cell death associated to glutathione depletion, lipid peroxidation and iron dependency could also be induced in plant cells by heat treatment. Unfortunately, the mediators and elements of the ferroptotic pathway have not been described yet. Our present results on Arabidopsis thaliana cell cultures suggest that acrolein, a lipid peroxide-derived reactive carbonyl species, is involved in plant ferroptosis-like cell death. The acrolein induced cell death could be mitigated by the known ferroptosis inhibitors such as Ferrostatin-1, Deferoxamine, α-Tocopherol, and glutathione. At the same time acrolein can be a mediator of ferroptosis-like cell death in plant cells since the known ferroptosis inducer RSL3 induced cell death could be mitigated by the acrolein scavenger carnosine. Finally, on the contrary to the caspase independent ferroptosis in human cells, we found that caspase-like activity can be involved in plant ferroptosis-like cell death.

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Plant UCPs are proved to take part in the fine-tuning of mitochondrial ROS generation. It has emerged that mitochondrion can be an important early source of intracellular ROS during plant-pathogen interaction thus plant UCPs must also play key role in this redox fine-tuning during the early phase of plant–pathogen interaction. On the contrary of this well-established assumption, the expression of plant UCPs and their activity has not been investigated in elicitor induced oxidative burst. Thus, the level of plant UCPs both at RNA and protein level and their activity was investigated and compared to AOX as a reference in Arabidopsis thaliana cells due to bacterial harpin treatments. Similar to the expression and activity of AOX, the transcript level of UCP4, UCP5 and the UCP activity increased due to harpin treatment and the consequential oxidative burst. The expression of UCP4 and UCP5 elevated 15-18-fold after 1 h of treatment, then the activity of UCP reached its maximal value at 4h of treatment. The quite rapid activation of UCP due to harpin treatment gives another possibility to fine tune the redox balance of plant cell, furthermore explains the earlier observed rapid decrease of mitochondrial membrane potential and consequent decrease of ATP synthesis after harpin treatment.

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