Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was ...not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation.
To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane‐bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC ...1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day–night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane‐bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.
An improved method of cell fractionation allowed the extraction of soluble (sNR) and membrane-associated
(mNR) forms of nitrate reductase (NR) from a dinoflagellate, even though in previous studies ...only mNR had been
found in these algae. Both activities were assayed in cell-free extracts of Peridinium gatunense from Lake Kinneret,
Israel, after disruption of the cells and differential centrifugation. In the cultures used, sNR showed much higher
NO3−-reducing activity. Only a low proportion, 2.5–3% of NR activity, was found to be associated with mNR.
Moreover, mNR comprised two forms as indicated by protein solubilization: a tightly membrane-bound and a
more weakly attached NR. Ascorbate inhibited all NR activities, but that of mNR recovered after its removal.
Polyvinyl pyrrolidone (PVP) and DTT also diminished sNR and mNR activities. For both enzymes, pH optima
(7.65) and temperature optima (13–25°C) were similar, and agreed with those for optimum growth of P. gatunense
both in culture and in the lake. The most efficient electron donor was NADH, though NADPH sustained low NR
activities. Carboxylic anions such as succinate and malate did not support any reduction of NO3−, nor did they
cause any stimulation of sNR or mNR activities. Both forms of NR showed a high affinity for their substrates:
Km was c. 10 μM for NO3−
and c. 5 μM for NADH. The high efficiency of NO3− assimilation by Peridinium seems
to be limited mainly by energy under otherwise optimal nutritional conditions and, at low nitrate concentrations,
the low Km may be one of the main reasons for the high competitivity of this alga in Lake Kinneret.
This text focuses on the most up-to-date developments in our understanding of how plants use light energy and fixed carbon to assimilate nitrate and ammonium into the organic compounds required for ...growth. From the partitioning of organic nitrogen within the photosynthetic apparatus, through the primary processes of reduction of nitrate and nitrite and the assimilation of ammonium and its cycling in photorespiration, the complex interactions inherent in the crosstalk between carbon and nitrogen assimilation are considered and exciting new developments such as nitric oxide production evaluated. Attention is paid throughout to the close co-ordination of photosynthetic and respiratory processes in nitrogen assimilation. Emerging concepts of the interdependence of chloroplasts and mitochondria are described, and essential communication, transport and signalling processes are highlighted. This is the first comprehensive treatise on photosynthetic nitrogen assimilation.
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