Nitrite reduces cytoplasmic acidosis under anoxia

I.G.L. Libourel, P.M. van Bodegom, M.D. Fricker, R.G. Ratcliffe

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The ameliorating effect of nitrate on the acidification of the cytoplasm during short-term anoxia was investigated in maize (Zea mays) root segments. Seedlings were grown in the presence or absence of nitrate, and changes in the cytoplasmic and vacuolar pH in response to the imposition of anoxia were measured by in vivo 31P nuclear magnetic resonance spectroscopy. Soluble ions and metabolites released to the suspending medium by the anoxic root segments were measured by high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy, and volatile metabolites were measured by gas chromatography and gas chromatography-mass spectrometry. The beneficial effect of nitrate on cytoplasmic pH regulation under anoxia occurred despite limited metabolism of nitrate under anoxia, and modest effects on the ions and metabolites, including fermentation end products, released from the anoxic root segments. Interestingly, exposing roots grown and treated in the absence of nitrate to micromolar levels of nitrite during anoxia had a beneficial effect on the cytoplasmic pH that was comparable to the effect observed for roots grown and treated in the presence of nitrate. It is argued that nitrate itself is not directly responsible for improved pH regulation under anoxia, contrary to the usual assumption, and that nitrite rather than nitrate should be the focus for further work on the beneficial effect of nitrate on flooding tolerance.
Although nitrate has a beneficial effect on plant survival under anoxia, the relative importance of the mechanisms by which this might be achieved is unclear (Gibbs and Greenway, 2003; Greenway and Gibbs, 2003; Stoimenova and Kaiser, 2004). Recent work has confirmed both the physiological significance of nitrate and/or nitrate reduction in moderating the impact of oxygen deprivation, and the difficulty of providing conclusive mechanistic explanations (Stoimenova et al., 2003; Allègre et al., 2004).

For example, transgenic tobacco (Nicotiana tabacum) plants lacking root nitrate reductase (NR) activity were more sensitive to root anoxia than wild-type control plants, but the metabolic phenotype of the transformants was unexpectedly complicated, with a substantial increase in fermentation under anoxia and a greater acidification of the cytoplasm (Stoimenova et al., 2003). It was concluded that the absence of NR activity in the transgenic roots did not limit the regeneration of NADH, and it was suggested that the beneficial effects of nitrate reduction might lie in a down-regulation of the metabolic rate under anoxia (Stoimenova et al., 2003). Similarly, experiments on hydroponically grown tomato (Lycopersicon esculentum) plants during the development of root anoxia showed that nitrate reduction was beneficial in preventing the onset of wilting and necrosis in the leaves, and that anoxia caused increases in NR activity and nitrite release to the growth medium (Allègre et al., 2004). The effects on NR activity and nitrite production, which were confirmed in parallel experiments on excised root systems (Morard et al., 2004), were in agreement with the current model for NR activation (Kaiser et al., 1999), but again it was not possible to draw firm conclusions about the protective effect of nitrate reduction on the plants. Thus, the impact of nitrate and nitrate reduction on anaerobic metabolism in plants is incompletely understood and merits further investigation.
One major area of uncertainty is the relationship between nitrate and pH regulation during oxygen deprivation. This link is important because cytoplasmic acidosis is a determinant of flooding intolerance (Roberts et al., 1984a), and exposure to nitrate was found to reduce the acidification of the cytoplasm in anoxic maize (Zea mays) root tips (Roberts et al., 1985). Since NR is reversibly activated at low pH (Kaiser and Brendle-Behnisch, 1995) and since nitrate reduction is proton consuming, it has been argued that nitrate reduction could form the basis for a biochemical pH-stat (Botrel et al., 1996; Botrel and Kaiser, 1997; Ratcliffe, 1999; Greenway and Gibbs, 2003). Unfortunately, this proposal is unsustainable because nitrate reduction generates protons when the only source of reducing power is glycolysis (Gerendás and Ratcliffe, 2002; Libourel, 2003). In agreement with this analysis, anaerobic metabolism was demonstrated to be more acidifying in wild-type tobacco roots capable of reducing nitrate to nitrite than in transformants lacking NR (Stoimenova et al., 2003). Thus, the reason for the beneficial effect of nitrate on pH regulation under anoxia is unknown.

In principle, the improvement in pH regulation could reflect either a direct effect of nitrate metabolism on cytoplasmic pH itself, or an indirect nitrate-induced change in carbon metabolism or ion transport with consequential changes in pH regulation (Libourel, 2003). To assess the contribution of these mechanisms, the response of maize root segments to short-term anoxia was examined in the presence and absence of nitrate by: (1) measuring changes in cytoplasmic and vacuolar pH using in vivo 31P NMR (Ratcliffe, 1997), and (2) profiling the soluble and volatile ions and metabolites released by anoxic root segments into the surrounding medium. NR is substrate inducible, and a comparison was made between: (1) roots grown on nitrate and subjected to anoxia in the presence of nitrate; (2) roots grown on chloride, switching to nitrate during the anoxic experiment; and (3) roots grown and treated with chloride only. The most striking difference between the root exudates turned out to be in the level of nitrite, and the subsequent demonstration that a low level of nitrite itself is sufficient to reduce the acidification of the cytoplasm under anoxia focuses attention on a primary role for nitrite rather than nitrate in the anaerobic response.
Original languageEnglish
Pages (from-to)1710-1717
Number of pages8
JournalPlant Physiology
Publication statusPublished - Dec 2006


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