The contribution of bacteroidal nitrate and nitrite reduction to the formation of nitrosylleghaemoglobin complexes in soybean root nodules

It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO(*)) in root nodules, although the sources of this NO(*) within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO(*) can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO(*) by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO(*) within nitrate-treated soybean nodules, electron paramagnetic resonance and UV-visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO(*) production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.