Physiological studies on a nonsymbiotic plant haemoglobin in a transgenic maize cell system
Nonsymbiotic haemoglobins are broadly present across the plant kingdom, however, the function of these proteins is unknown. Cultured maize cells have been transformed to constitutively express a barley haemoglobin gene in either the sense (HB$\sp+$) or antisense (HB$\sp-$) orientation, Haemoglobin protein expression in the transformed cell lines was correspondingly higher or lower than in wild type cells under normal atmospheric conditions. Limiting oxygen availability by placing the cells in a nitrogen atmosphere for 12 hours had little effect on the energy status of cells constitutively expressing haemoglobin, but had a pronounced effect on both wild type and HB$\sp-$ cells, where ATP levels declined by 27% and 61% respectively. Total adenylates in these cells were approximately 35% lower than in HB$\sp+$ cells. Energy charge was relatively unaffected by the treatment in HB$\sp+$ and wild type cells, but was reduced from 0.91 to 0.73 in HB$\sp-$ cells suggesting that the latter were incapable of maintaining their energy status under the low oxygen regime. Treatment of the cells grown in an air atmosphere with electron transport inhibitor, antimycin A gave essentially the same results. The presence of haemoglobin enhanced oxygen uptake by the cells under conditions of low oxygen availability. It is suggested that nonsymbiotic haemoglobins act in plants to maintain the energy status of cells in low oxygen environments and that they accomplish this effect by promoting glycolytic flux through NADH oxidation, resulting in increased substrate level phosphorylation. Hypoxic acclimation of plants is an example of this effect in nature. Nonsymbiotic haemoglobins are likely ancestors of an early form of haemoglobin that sequestered oxygen in low oxygen environments, providing a source of oxygen to oxidize NADH to provide ATP for cell growth and development.