Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase activity in leaf extracts: What limits nitrate reduction in situ?

Nitrate reductase (NR) activity in spinach leaf extracts prepared in the pr esence of a protein phosphatase inhibitor (50 mu M cantharidine) was measur ed in the presence of Mg2+ (NRact) or EDTA (NRmax), under substrate saturat ion. These in-vitro activities were compared with nitrate reduction rates i n leaves from nitrate-sufficient plants. Spinach leaves containing up to 60 mu mol nitrate per g fresh weight were illuminated in air with their petio le in water. Their nitrate content decreased with rime, permitting an estim ation of nitrate reduction in situ. The initial rates (1-2 h) of nitrate co nsumption were usually lower than NRact, and with longer illumination time (4 h) the discrepancy grew even larger. When leaves were fed through their petiole with 30 mM nitrate, initial in-situ reduction rates calculated from nitrate uptake and consumption were still lower than NRact. However, nitra te feeding through the petiole maintained the in situ-nitrate reduction rat e for a longer time. Initial rates of nitrate reduction in situ only matche d NRact when leaves were illuminated in 5% CO2. In CO2-free air or in the d ark, both NRact and in-situ nitrate reduction decreased, but NRact still ex ceeded in-situ reduction. More extremely, under anoxia or after feeding 5-a mino-4-imidazole carboxyamide ribonucleoside in the dark, NR was activated to the high light level; yet in spite of that, nitrate reduction in the lea f remained very low. It was examined whether the standard assay for NRact w ould overestimate the ia-situ rates due to a dissociation of the inactive p hospho-NR-14-3-3 complex after extraction and dilution, but no evidence for that was found. In-situ NR obviously operates below substrate saturation, except in the light at high ambient CO2. It is suggested that in the short term (2 h), nitrate reduction in situ is mainly limited by cytosolic NADH, and cytosolic nitrate becomes limiting only after the vacuolar nitrate pool has been partially emptied.