Nitric oxide in plants: the roles of ascorbate and hemoglobin

Abstract

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.

Figure 1. Pertinent chemical reactions of AA, hemoglobin, and NO.

A) Ascorbic acid (AA) can lose an electron to become monodehydroascorbate (MDHA) radical, which can lose an electron to become dehydroascorbate (DHA). B) The nitric oxide dioxygenase (NOD) reaction; oxy hemoglobins (Hb) will react with NO to make nitrate and ferric Hb. If the ferric Hb is reduced (in this case by AA), it will bind oxygen (if present) to start the reaction again. C) The AA/glutathione cycle in plants is associated with scavenging of peroxide, superoxide, and hydroxyl radical, and is hypothesized to reduce nsHbs in support of NOD.

Figure 2. nsHb reduction by AA, DHA, and MDHAR.

A) ferric rice nsHb (5 µM) was mixed with 3 mM AA in air-saturated buffer. The absorbance changes in the visible and B) Soret regions are associated with nsHb reduction and oxygen binding. C) Time courses for nsHb reduction by various concentrations of AA. D) The rate constants for AA reduction of nsHb and Mb (5 µM) are plotted as a function of AA concentration, which have slopes equal to the bimolecular rate constants for reduction of nsHb.

Figure 3. EPR measurements of MDHA radical in solutions of AA (A), and produced from AA reduction of nsHb (B).

A) Freshly dissolved 3 mM AA solutions contain MDHA, which decays to a lower equilibrium level over several hours. B) Addition of ferric nsHb to AA increases the EPR signal associated with MDHA.

Figure 4. The effect of MDHAR on AA reduction of nsHb.

A) Recombinant rice MDHAR was purified and quantified based on its flavin absorbance spectrum. B) The EPR signal associated with MDHA was used to demonstrate the activity of AAOx (which increases the MDHA signal in the presence of AA), and MDHAR (which decreases MDHA in the presence of NADH). These data also show that NO reacts directly with MDHA. C) MDHAR oxidizes NADH in the presence of MDHA (produced from AA and AAOx, as in (B)). D) Time courses for nsHb reduction by AA in the absence and present of MDHAR and NADH are nearly identical. E) When nsHb concentration is greater than AA, reduction is very slow, and slightly faster in the presence of MDHAR and NADH.

Figure 5. NO scavenging by AA, nsHb, and MDHAR.

A) NO scavenging by buffer, AA, and DHA are compared under aerobic and hypoxic (0.1 µM [O₂], inside the yellow circle) conditions. B) NO scavenging with MDHAR and AAOx under aerobic and hypoxic (inside the yellow circle) conditions. The curves for NO alone are black, and the presence of AA (red), MDAHR and AA (blue), and AAOx (green) are indicated by color. A dashed line indicates the presence of NADH (0.2 mM) in that reaction condition. C) NO scavenging by nsHb, with and without MDHAR are compared to that facilitated with reduction of nsHb by ferredoxin reductase (Fdr, green dashed line with filled circles, indicating Fdr and NADH are present along with nsHb). D) An expanded view of aerobic NO scavenging demonstrates the subtle effects of AA and DHA.

Figure 6. Production of NO from nitrite and AA.

NO production from solutions of nitrite (10 mM unless otherwise indicated) were stimulated by addition of AA at pH values ranging from 6 to 7. A) NO production is measurable at pH 6.75 and increases with decreasing pH. When 5 µM nsHb is included (red traces) there is a decrease in the level NO proportional to the Hb concentration. B) The amount of NO produced is directly related to nitrite concentration, as is evident from the high levels produced even at pH 7 when nitrite concentration is very high.