Review article: free radicals, antioxidants and human disease: where are we now?

This survey emphasizes the possibilities of isozymes as marker genes in solving a variety of problems important to population geneticists, evolutionists, systematists, and plant breeders.

Isozymes in plant biology

The ability to observe allelic variation at isozyme loci has revolutionized research in the fields of biochemical genetics, population genetics, and evolution. This variation, called allozymic polymorphism, has been used in plants to examine genetic processes at every stage of the life cycle and to ascertain genetic diversity in all major crops as well as many other species. Yet the potential for using allozymes as genetic markers was not immediately predicted when isozyme variability was initially described (Hunter and Marker, 1957; Markert and Moller, 1959). In fact, the extent and prevalence of allozyme polymorphism was a rather disconcerting surprise to evolutionary biologists. Classical evolutionary theory had predicted that the most “efficient” form of an enzyme should, over time, become predominant in isolated populations, with an occasional rare allele produced through mutation. The discovery in many populations of relatively high levels of polymorphism at isozyme loci has forced a major reconsideration of evolutionary theory (Kimura and Crow, 1964; Koehn et al., 1983; Kimura, 1983).

Genetics of Plant Isozymes

Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide.

Treatment of Arabidopsis cell culture for 16 h with H2O2, menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H2O2/menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H2O2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O2 consumption by mitochondria was significantly decreased by H2O2 treatment.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2002.01474.x

The impact of oxidative stress on Arabidopsis mitochondria.

Preincubation of potato (Solanum tuberosum) tuber mitochondria with 300 microM-H2O2 for 10 min nearly stopped the State 3 rate of citrate oxidation. Addition of isocitrate resulted in resumption of O2 uptake. The State 3 rates of succinate, external NADH and 2-oxoglutarate oxidation were unaffected by H2O2 over the dose range 50-500 microM. Preincubation of mitochondria with 300 microM-H2O2 for 5 min unmasked in the matrix space a paramagnetic signal with a peak at a g value of approx. 2.03. Aconitase was purified over 135-fold to a specific activity of 32 mumol/min per mg (with isocitrate as substrate) from the matrix of potato tuber mitochondria. The native enzyme was composed of a single polypeptide chain (molecular mass 90 kDa). Incubation of purified aconitase with small amounts of H2O2 caused the build up of a paramagnetic 3Fe cluster with a low-field maximum of g = 2.03 leading to a progressive inhibition of aconitase activity. The results show that aconitase present in the matrix space was the major intramitochondrial target for inactivation by H2O2.

Rapid inactivation of plant aconitase by hydrogen peroxide. Biochem J

Protoplasts obtained from sycamore (Acer pseudoplatanus) cell suspensions were found to be highly intact. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured leaving the fragile amyloplasts largely intact. Aconitase hydratase (citrate [isocitrate] hydro-lyase, EC 4.2.1.3) activity of sycamore cells was associated with two protein fractions, one present in the cytosol while the second is of mitochondrial origin. Chromatography on DEAE-trisacryl did not separate the aconitase hydratase isoenzymes. EPR studies established that both isoenzymes exhibited an EPR signal at g = 2.03 once oxidized.

Characterization of a Cytosolic Aconitase in Higher Plant Cells

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.

http://www.plantphysiol.org/content/plantphysiol/81/1/247.full.pdf

Jacques Gaillard

Papers

Rapid inactivation of plant aconitase by hydrogen peroxide. Biochem J

Characterization of a Cytosolic Aconitase in Higher Plant Cells

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria.