Regulation of NADH supply for nitrate reduction in green plants via photosynthesis and mitochondrial respiration

Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO2 production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity.done. Wherever preincubation was involved, the excised leaves were incubated with different treatment solutions for 3 h under light (800 ,uE m-2 S-') at 30°C. RESULTS AND DISCUSSIONGlycine enhanced nitrate reduction in barley leaves when included in the in vivo assay medium (Fig. 1). The maximum enhancement was attained at 10 mm concentration. Three possible explanations could be attributed to explain this enhancement: a. possible induction ofNR by the amino acid; b. increased stability of the enzyme in the presence of ammonium (13) released during glycine oxidation; c. increased supply of reducing power to the enzyme. It has been observed by many workers that amino acids either repress the synthesis or reduce the activity of NR (10). We observed no considerable change in NR activity by using a wide range of amino acids other than glycine in the NR'(EC

Section: Methodssupporting

confidence: 66%

Glycine Supports in Vivo Reduction of Nitrate in Barley Leaves

Kumar

Nair²,

Abrol³

1988

“…Although the role of light on nitrate assimilation has been studied extensively, controversy continues as to whether nitrate reduction normally occurs in darkness. Nitrate reduction has been reported in tissue slices in darkness under anaerobic conditions, detected as nitrite accumulation in the medium (13,(17)(18)(19). Under aerobic conditions in the dark, however, nitrite does not accumulate (this report, 3,13,17,19).…”

Section: Resultsmentioning

confidence: 59%

“…That problem can be resolved only by following each of the processes involved: nitrate uptake, accumulation, and reduction or at least the disappearance of substrate (nitrate), and the appearance and/or disappearance of the product (nitrite). Sawhney et al (18,19) Our results clearly show that nitrate reduction in intact barley seedlings under aerobic conditions occurs at linear rates in both light and darkness (Fig. 1).…”

Section: Resultsmentioning

confidence: 67%

“…An obligatory requirement of light for nitrate assimilation has been reported for several species (6,18,19), whereas others report nitrate reduction in darkness in tissue slices or detached leaves of many species under both anaerobic (3,13,(17)(18)(19) and aerobic (12,13) conditions. Sawhney et aL (18) recently proposed that reduction of nitrate in light only avoids the accumulation of toxic levels of nitrite in the dark. In contrast, Jones and Sheard (12) observed that both nitrate and nitrite are reduced in leaf slices in darkness.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Influence of Light and Ambient Carbon Dioxide Concentration on Nitrate Assimilation by Intact Barley Seedlings

Aslam¹,

Huffaker²,

Rains³

et al. 1979

136

The influence of light, dark, and ambient CO2 on nitrate assimilation in 8-to 9-day-old barley seedlngs was studied. To develop the photosynthetic apparatus fully, the seedings were grown in nitrogen-free Hoagland solution for 5 days in darkness followed by 3 days in continuous ight.The It is well established that light energy is intimately involved in the assimilation of nitrate (1,(4)(5)(6)24). The influence of light is not well understood, however, and reports are contradictory. An obligatory requirement of light for nitrate assimilation has been reported for several species (6,18,19), whereas others report nitrate reduction in darkness in tissue slices or detached leaves of many species under both anaerobic (3,13,(17)(18)(19) and aerobic (12, 13) conditions. Sawhney et aL (18) recently proposed that reduction of nitrate in light only avoids the accumulation of toxic levels of nitrite in the dark. In contrast, Jones and Sheard (12) and placed in the dark at room temperature. On the 6th day, the seedlings were transferred to aerated one-fourth-strength Hoagland solution (9) lacking nitrogen and placed in continous light of 500 ,uE m-2 s-1 for 3 more days at 25 C and 70 to 75% RH to develop the photosynthetic apparatus (10). When carbohydratedeficient seedlings were used, they were given 3 days of light treatment and then placed in darkness at 25 C and 70 to 75% RH for the desired period (24-48 h).Nitrate and Nitrite Uptake. Uptake of nitrate and nitrite was measured as the amounts disappearing from the substrate solution with time. Ten seedlings per treatment (each treatment replicated twice and each experiment repeated two times) were placed in 140 ml of one-fourth-strength Hoagland solution containing 1.0 mm KNO3 or 1.0 mm NaNO2 and 5.0 mm CaSO4. The initial pH of the solutions was 5.8. The solutions were renewed once after a 12-h absorption period. By this time, nitrate concentration from the uptake medium was reduced to about 0.5 mm. Previous studies in this laboratory have shown that uptake rates between 0.5 and 1.0 mm nitrate were constant (7). When excised roots were used, they were excised at the scutellar node and submerged in the uptake medium for the desired periods. Two g of excised roots per treatment were used. All solutions were aerated during uptake.The addition of 50 jig/ml chloramphenicol to uptake solutions had no effect on the results showing that bacterial contamination was not a problem.To study the effect of CO2 on nitrate uptake, the seedlings were placed in a 15-liter Plexiglas chamber. The light intensity inside the chamber at the top of the seedling canopy was 450 ,uE m-2 s-'.Normal or CO2-free air was passed through the chamber at 2 liters/min, giving about eight exchanges/h, with a positive pressure inside the chamber. The treatment solutions were aerated with the same gas. The air from the Plexiglas chamber was passed through a cold bath and recirculated back into the chamber at 6 liters/min. This maintained temperature and RH at about the same level as in the growth cham...

“…It was earlier observed (13,14) oxidase by CO, which in turn was a reflection of the reduced state of Cyt a3 during steady-state respiration. This technique was earlier used to examine CO-sensitivity of Cyt c oxidase in the leaves of wheat (9), pearl millet (7), and sorghum (8).…”

mentioning

confidence: 99%

Differential Carbon Monoxide Sensitivity of Cytochrome c Oxidase in the Leaves of C₃ and C₄ Plants

Naik

Dhage²,

Munjal³

et al. 1992

CO sensitivity of cytochrome a3 in the leaves of a number of C3 and C4 plants was monitored by the nitrate reductase assay under differing CO to 02 ratios. All the C3 plants were relatively insensitive to CO and required a high CO to 02 ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to 02 ratios of 10 or less. On the other hand, the leaves of all C4 plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to 02 ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from me sophyll or bundle sheath cells or both, lacked tight respiratory control.Cyt c oxidase (EC 1.9.3.1), the multiunit enzyme complex consisting of Cyt a and Cyt a3 and associated CuA and CuB components, is the terminal oxidase in the mitochondrial electron transfer chain and contains site 3 of oxidative phosphorylation (16). CO is a specific inhibitor of the enzyme, as it forms a complex with reduced Cyt a3 (1, 3). In mammalian mitochondria, this complex is considered to be stable in the dark but can be photodissociated after exposure to a flash of light with absorption maxima at 430 and 590 nm (2). We had recently observed that in a number of plant species, CO can be replaced from the active site in complete darkness when excess 02 is supplied (10). Pollok et al. (12) oxidase by CO, which in turn was a reflection of the reduced state of Cyt a3 during steady-state respiration. This technique was earlier used to examine CO-sensitivity of Cyt c oxidase in the leaves of wheat (9), pearl millet (7), and sorghum (8).In these studies, the leaves were exposed to CO briefly for 1 min and then incubated under aerobic conditions for the determination of nitrite accumulation in vivo. Hence, the relative concentrations of CO and 02 during incubation were not known with certainty. We have now examined a large number of C3 and C4 plants, exposed to precisely defined CO to 02 ratios during the course of incubation. We now report that there are distinct differences between C3 and C4 plants with regard to CO-sensitivity of Cyt c oxidase. Thus, the leaves of all C4 plants tested are highly sensitive even at low CO to 02 ratios, whereas in C3 plants, considerably higher CO concentrations are required to inhibit Cyt c oxidase.Because the redox state of Cyt a3 during steady-state respiration probably depends on the tightness of coupling of oxidative phosphorylation at site 3, the results suggest possible differences in this regard between the leaves of C3 and C4 plants.