In the course of nitric oxide (NO) scavenging, hemoglobin (Hb) turnover is linked to antioxidant metabolism and affects the cellular redox level. The influence of Hb presence on the ascorbate-glutathione cycle enzymes and the levels of H(2)O(2) and ascorbate was investigated in alfalfa root cultures transformed to over-express (Hb+) or down-regulate (Hb-) class-1 Hb. Hb+ lines had substantially increased ascorbate levels as well as elevated monodehydroascorbate reductase and ascorbate peroxidase activities. Hb- lines showed significant increases in dehydroascorbate reductase and glutathione reductase activities. The observed changes in ascorbate and ascorbate-glutathione cycle enzymes were pronounced both at high (40 kPa) and low (3 kPa) O(2) pressures. Hb- lines had significantly reduced levels of the NO- and H(2)O(2)-sensitive enzyme, aconitase, as compared to Hb+ lines. This reduced activity was likely due the higher levels of NO in Hb- lines, as treatment of plant extracts with the NO-donor DEANO also affected aconitase activity. The H(2)O(2) levels were not significantly different amongst the lines and showed no variation with change in oxygen partial pressure. In conclusion, the expression of class-1 Hb improves the antioxidant status through increased ascorbate levels and increased activity of enzymes involved in H(2)O(2) removal.