An increase in the concentration of lipid hydroperoxides was observed during soybean nodule aging. The concentration of H2O2 itself also increased in the same period of nodule development. The H2O2 increase paralleled that for peroxides (Evans et al. 1999). H2O2 production was detected in ultrathin sections of senescent soybean nodules as an electron-dense deposit stained with cerium chloride (Bestwick et al. 1997). A significant labeling was detected in the cell walls and around the peribacteroid membranes, which are amongst the first structures to be degraded during senescence. No H2O2 was detected in the non-infected cells. H2O2 was also evidenced in the senescing zone (zone IV) of alfalfa nodules, whereas it could not be detected in the nitrogen-fixing zone (zone III).
H2O2 is known to react with leghemoglobin in vitro, to generate modified hemoproteins which most probably occur from an intramolecular cross-link of a phenoxyl radical (involving a tyrosine residue) to the heme group (Moreau et al. 1996). One of these species is green in color and has a pi of 5.45. This species was detected in senescing nodules but not in extracts from four-week-old nodules (Mathieu et al. 1997). Electron paramagnetic resonance (EPR) spectroscopy has also been employed to examine the nature of radicals present in these organs. When EPR spectra of intact and largely senescent nodules were recorded at low temperature, typical signals were obtained. Exposure of young and mature nodules to oxidative stress, in the form of exogenous H2O2 addition, resulted in changes in the EPR spectra with the appearance of absorptions similar to those from untreated senescent nodules. In particular, one signal is believed to be due to an altered form of leghemoglobin (Mathieu et al. 1998).
The catalytic iron content greatly increased during nodule development and the increase appeared to be linear with age. When the peroxide level was plotted versus iron content, a linear correlation was obtained (Evans et al. 1999). Proteins are also damaged, including generation of carbonyls groups on certain amino acid side chains, by oxidative processes. There was also a rise in carbonyls with nodule age. Concentrations of xanthine and hypoxanthine, deamination products of the purines adenine and guanine, also increased during senescence, pointing to the existence of some DNA damage during nodule aging (Evans et al. 1999). All these results are consistent with the development of an oxidative stress during nodule senescence.
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