Redox Enzymes of Red Beetroot Vacuoles (Beta vulgaris L.)

Years of research have shown that some of the redox elements (enzymes, coenzymes, and co-substrate) are isolated from each other kinetic and spatial manner (compartmentalization) in the eukaryotic cells. The redox elements forming the "highly" and "widely" specialized redox system are found in all cell structures: mitochondria, plastids, peroxisomes, apoplast, nucleus etc. In recent years the active involvement of the central vacuole in the maintenance of the plant cell redox homeostasis is discussed, actually the information about the vacuolar redox system is very small. The high-priority redox processes and "redox-specialization" of the vacuolar compartment are not known. We have begun a study of red beet-root vacuole redox systems (Beta vulgaris L.) and have identified redox enzymes such as: phenol peroxidase (EC 1.11.1.7), superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.8.1.7). This paper presents some of the characteristics of these enzymes and considers the probable ways of their functioning in vacuolar redox chains

Glutathione Reductase of Vacuole. Comparison of Glutathione Reductase Activity of Vacuole and Tissue Extract of Red Beet Root (Beta vulgaris L.)

Glutathione reductase (GR, EC 1.8.1.7) is the enzyme that reduces oxidized glutathione (GSSG) and thus regulates the redox state of glutathione (GSH/GSSG). GR has been studied in most plants. This enzyme has been identified in chloroplasts and cytosol, so these cellular compartments are considered to be the main place of the enzyme localization. In the same time, just a little is known about GR vacuoles. There are no conclusive evidences to prove the presence or absence of this enzyme in the vacuoles. GR activity was found in the vacuoles of red beet root cells (Beta vulgaris L.). The level of activity, the optimum pH and isoenzyme composition of GR were compared in the vacuoles and tissue extract of beet root. Vacuolar GR activity was quite high, it was 1.5-2 times higher than the activity of the tissue extract. Enzyme pH optimum of all the objects were identical. pH-optimum depend on the pyridine nucleotide nature: pH 7.0-8.0 was an optimal range with NADPH; pH 5.0 – with NADH. GR activity of the vacuoles and tissue extracts decreased in the presence of a noncompetitive inhibitor 1-chloro-2.4-dinitrobenzene (CDNB), indicating the specificity of this enzymatic reaction. Two bands with glutathione reductase activity have been identified in the vacuoles and tissue extracts using zymography method to determine the enzymatic activity in PAAG after electrophoresis of proteins. Belonging to the GR isoforms of these bands was confirmed by enzyme immunoassay (Western blotting). The electric mobility of isoforms of the study objects did not differ significantly. It is concluded that the biochemical characteristics of vacuolar glutathione reductase were substantially identical to the biochemical characteristics of other localization GR

The Effect of Herbicides on Hydrogen Peroxide Generation in Isolated Vacuoles of Red Beet Root (Beta vulgaris L.)

Influence of herbicides on the hydrogen peroxide generation in vacuolar extracts of red beet root (Beta vulgaris L.) was investigated. Belonging to different chemical classes of herbicide compounds have been used. Herbicides differ from each other in the mechanism of effects on plants. Clopyralid (aromatic acid herbicide, derivative of picolinic acid) and 2.4-D (phenoxyacetic herbicide), characterized by hormone-like effects, contributed to the formation of H2O2 in vacuolar extracts. Fluorodifen (nitrophenyl ether herbicide) and diuron (urea herbicide) also have increased contents H2O2. These compounds inhibit the electron transport, photosynthesis, and photorespiration in sensitive plants. Herbicidal effect of glyphosate (organophosphorus herbicide) is due to the inhibition of amino acid synthesis in plant cells. Glyphosate did not affect the content of H2O2 in vacuolar extracts. Herbicide dependent H2O2-generation did not occur with oxidoreductase inhibitors, potassium cyanide and sodium azide. The results suggest that the formation of ROS in the vacuoles due to activity of oxidoreductases, which could interact with herbicides

Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

The glutathione of the red beetroot vacuoles (Beta vulgaris L.) was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT); the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB); the high-performance liquid chromatography (HPLC). The content of reduced (GSH) and oxidized glutathione (GSSG) differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal) – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione