NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin.

Transgenic alfalfa ( Medicago sativa L.) root cultures expressing sense and antisense barley ( Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000- g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7-8 and a strong affinity to NADH and NADPH ( K(m) 3 microM for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35+/-4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.

Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress.

Transgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb+) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb-) experienced 30-70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb+ lines did not significantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb- lines were significantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb- line was 2.5-fold higher than that in the Hb+ line. Treatment of transgenic root lines under 40% oxygen with NO resulted in significant declines in the ATP levels and ATP/ADP ratio of an Hb- line and the control line, with no significant change in an Hb+ line. The root cell structure of an Hb- line showed evidence of cell breakdown under hypoxic growth, whereas an Hb+ line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.