Different diurnal cycles of expression of two nitrate reductase transcripts in tobacco roots.

In roots and leaves of tobacco (Nicotiana tabacum cv. Samsun) three functional transcripts (3.6 kb, 3.1 kb, and 1.8 kb) were found to at least partly represent nitrate reductase mRNA. With specific probes for the transcripts of the different domains of nitrate reductase it was shown that the smallest transcript was shortened in the region coding for the flavin adenine dinucleotide domain and might be the transcript coding for plasma-membrane-bound nitrate reductase. The expression of the 3.1 kb and 1.8 kb transcripts in roots was differently regulated during the day-night cycle with the maximum amount of the 3.1 kb transcript in the middle and of the 1.8 kb transcript at the end of the light period.

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite.

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.

Regulation by amino acids of photorespiratory ammonia and glycolate release from ankistrodesmus in the presence of methionine sulfoximine.

Methionine sulfoximine induced release of ammonia from illuminated cells of Ankistrodesmus braunii (Naegeli) Brunnth, in normal air, but less in air enriched to 3% CO(2). In normal air, methionine sulfoximine also induced glycolate release. Addition of either glutamate, glycine, or serine suppressed glycolate release, whereas glutamate and glycine at the same time stimulated ammonia release. The results indicate that inhibition of glutamine synthetase and thereby inhibition of photorespiratory nitrogen cycling restricts the sink capacity for glycolate in the photorespiratory carbon cycle. An external supply of glutamate, glycine, or serine seems to stimulate glyoxylate transamination and thus partly restores the sink capacity. Calculations of total glycolate formation rates in air from glycolate and ammonia release rates in the presence of methionine sulfoximine and glutamate revealed values of approximately 20 micromoles glycolate per milligram chlorophyll per hour on the average. Similar calculations led to an estimated rate of photorespiratory ammonia release in air, in the absence of methionine sulfoximine, of about 10 micromoles per milligram chlorophyll per hour on the average, a value comparable to the primary nitrogen assimilation rate of 8 micromoles per milligram chlorophyll per hour.

Effect of Glucose and CO(2) on Nitrate Uptake and Coupled OH Flux in Ankistrodesmus braunii.

In Ankistrodesmus braunii, in the absence of CO(2), i.e. in CO(2)-free air or N(2), photosynthetic nitrate uptake and nitrate reduction were inhibited, especially at low pH. Under such conditions, glucose stimulated nitrate uptake and reduction to almost the same level in the pH range between 6 and 8.5. CO(2) at 0.03% effected an intermediate pH dependence of nitrate uptake; saturating CO(2) concentration (more than 1%) eliminated the pH dependence, as did glucose, but the rates were enhanced compared with glucose. Glucose and, even more, CO(2), drastically reduced the release of nitrite and ammonia to the medium, the stoichiometry between alkalinization of the medium and nitrate uptake (OH(-)/NO(3) (-)) approached 1.Due to the lack of storage vacuoles in Ankistrodesmus, nitrate uptake and nitrate reduction were closely coupled processes whose experimental separation is difficult. The relieving effect of glucose and CO(2) suggests a carrier-mediated nitrate uptake which is more limiting than nitrate reduction and is sensitive to low pH, but which is stabilized by some intermediate originating from an active carbon metabolism.

Stoichiometry between photosynthetic nitrate reduction and alkalinisation by Ankistrodesmus braunii in vivo.

The uptake of nitrate or nitrite in the light, the release of nitrite and ammonia, and the corresponding alkalinisation of the medium were measured in synchronous Ankistrodesmus braunii (Naeg.) Brunnth. The increase in the OH(-) concentration in the medium reflects a stoichiometric ratio between OH(-) and NO3 (-) of 1.3-1.8 in air, reaching almost 2.0 in CO2-free air or nitrogen. At low CO2 concentrations a large proportion of the nitrogen taken up as nitrate is released as ammonia, much less as nitrite. The stoichiometry of alkalinisation and NO3 (-) or NO2 (-) uptake can be quantitatively explained by assuming: 1) a counter-transport, at a ratio of 1:1, of OH(-) against NO3 (-) at the plasmalemma and of OH(-) against NO2 (-) at the chloroplast envelope, and 2) a co-transport of 1:1 of OH(-) and NH4 (+) to the medium through both membranes. The first OH(-) required is formed by proton consumption in nitrite reduction, the second OH(-) by proton consumption in the formation of NH4 (+) ions. Transport of K(+), Na(+) and Ca(2+) is not or only scarcely involved. This proposed transport system could provide charge equilibrium between inside and outside the cells and could enable the cells to avoid nternal pH changes in nitrate and nitrite reduction.

[The nitrate- and nitrite-dependent O2-evolution in N 2 by Ankistrodesmus braunii]. / Die nitrat- und nitritabhängige photosynthetische O2-Entwicklung in N 2 bei Ankistrodesmus braunii.

O2-evolution in N2 by young cells of synchronous Ankistrodesmus braunii was measured manometrically in the presence and in the absence of nitrate or nitrite. Nitrate-starved cells produce O2 as a function of nitrate concentration with an optimum at 10 mM nitrate. The optimum rates are strongly dependent upon the pH of the medium culminating at pH 8.0.Nitrite excretion is initially slow and has its optimum at the same nitrate concentration as O2-evolution. It is slower under anaerobic conditions than in the presence of CO2 and slower at pH 5.6 than at pH 8.0. At pH 8.0 in N2 an accumulation of nitrite starts 40 to 100 min after the addition of nitrate to the algae.O2-evolution is faster with nitrite than with nitrate. No optimum curve is observed at pH 8.0 with various concentrations of nitrite in the medium; however, at pH 5.6 a distinct peak is found at 3 mM nitrite. This peak is found for a fast short-time reaction as well as for the following low rates of O2-evolution.The uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP) equally inhibits nitrate- and nitrite-dependent O2-evolution and the incorporation of (32)P into cellular phosphate compounds. There is no indication of uncoupling in vivo of nitrite reduction which is completely independent of ATP in vitro. The inhibition of the nitrate-dependent O2-evolution by high concentrations of nitrate cannot be explained by an accumulation of products such as ammonia or nitrite.

[The influence of CO2 and pH on (32)P-labelling of polyphosphates and organic phosphates in Ankistrodesmus braunii in the light]. / Der Einfluß von CO2 und pH auf die (32)P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht.

The effect of CO2 on the (32)P-labelling of polyphosphates and acid-soluble organic phosphates is studied in synchronously grown cultures of the green alga Ankistrodesmus braunii, using trichloroacetic acid treatment and acid hydrolysis for the fractionation of the phosphorus compounds.Three per cent CO2 in nitrogen causes an inhibition of the labelling of polyphosphates but a marked increase of (32)P in organic phosphates, whereas oxygen (CO2-free air) produces the reverse effect. Polyphosphates and ATP are the fractions most stimulated by O2, while stable organic phosphates show the strongest inhibition. Labelling of nucleic acids is relatively indifferent to both oxygen and CO2. Three per cent CO2 in air causes the same distribution of (32)P-labelling as 3 per cent CO2 in N2. (32)P-labelling is strongly dependent on the pH of the medium. In the absence of CO2, polyphosphate labelling is highest in the acidic range, whereas organic phosphates and ATP show optimum labelling and the highest percentage of the total (32)P in the alkaline pH range. The effect of CO2 is strongest between pH 5 and 6, that of oxygen between pH 8 and 9. Apparently the pH of the medium exerts a considerable influence upon the phosphate metabolism inside the cells.Increasing concentration of CO2 lead to the same change of (32)P-labelling in nitrogen as in air and to saturation at about 1 per cent CO2 under the conditions used. The curves are in good agreement with those of O2-evolution at increasing concentrations of CO2, but they show completely different rates.Young cells respond to CO2 and O2 differently from cells in the photosynthetically most active stage. In young cells both gasses are less effective.The effect of CO2 is explained by a strong increase in noncyclic photophosphorylation which can proceed only slowly in N2. ATP-consumption connected with high rates of CO2-fixation may be the reason for the low rates of (32)P-labelling in the polyphosphate fraction when CO2 is present. The influence of external pH on (32)P-labelling is partly due to the pH-dependence of phosphate uptake, but the different response of several fractions to the pH of the medium suggests that the pH of the cytoplasm and possibly even the pH of the interior of the chloroplasts is affected by the external pH. The effect of O2 in the absence of CO2 or at low CO2-concentrations is explained by the well-known inhibition of photosynthesis by oxygen. Increasing concentrations of CO2 reverse this inhibition and correspondingly change the distribution of (32)P between the phosphate fractions. The change in sensitivity to CO2 and O2 with the cell age is consistent with the change in the rates of maximum photosynthetic CO2-fixation.

[Nitrate-dependent noncyclic photophosphorylation in Ankistrodesmus braunii in the absence of CO2 and O 2]. / Nitratabhängige nichtcyclische Photophosphorylierung bei Ankistrodesmus braunii in Abwesenheit von CO2 und O 2.

Manometric measurements show that oxygen evolution proceeds in synchronised cells of Ankistrodesmus braunii even in an atmosphere of pure nitrogen. In this case the slow oxygen evolution is dependent on the presence of nitrate (Table 1). Light saturation is found at a low light intensity at pH 5.6, at a higher light intensity at pH 8.0 (Fig. 1). The light saturation curves are in good agreement with those of (32)P-labelling in Ankistrodesmus under the same conditions (Fig. 2).DCMU inhibition in N2 of both O2-evolution and (32)P-labelling begins only at a DCMU concentration of 5×10(-7)M or more. Complete inhibition of O2-evolution is reached only at 10(-5)M (Fig.3). In (32)P-labelling a variable percentage is still left uninhibited at 10(-5) M DCMU (Fig. 4, Table 2), which is at least partly due to cyclic photophsphorylation. Nitrate starvation for several hours causes a considerable decrease in O2-evolution and also in the sensitivity to those high concentrations of DCMU (Fig. 5), but it leads to a sensitivity to antimycin A not observed under normal conditions (Table 3). The effects of nitrate starvation thus become comparable to those of far-red light, under which noncyclic electron transport is slow or completely prevented.The inhibition by DCMU of electron transport in photosystem II is also estimated by measuring the increase in fluorescence at 684 nm in air containing additional CO2. This fluorescence is saturated only at 10(-5)M DCMU and shows that a certain percentage of photosystem II remains uninhibited at 5×10(-7)M (Fig. 6), a concentration found to be almost ineffective in inhibiting O2-evolution and (32)P-labelling in an N2-atmosphere.The results indicate that in synchronised cells of Ankistrodesmus noncyclic electron flow and noncyclic photophosphorylation can proceed in an atmosphere of pure nitrogen if nitrate is available as the electron acceptor. In this case noncyclic photophosphorylation, inspite of its low rates, still dominates over cyclic photphosphorylation. At low pH, when nitrate reduction is slow, cyclic photophosphorylation accounts for a greater part of the total phosphorylation than at high pH. Thus in the absence of CO2 and O2 cyclic photophosphorylation can be regarded as the main process of ATP formation only after nitrate starvation, in far-red light or in the presence of high concentrations of DCMU.Inhibition by DCMU, though very efficient under conditions of high photosynthetic activity, becomes rate-limiting only if the electron transport is so far reduced by DCMU that the remaining rate is of the same order as the low rate of the control or less. Therefore high concentrations of DCMU are required for the inhibition of low rates of noncyclic photophosphorylation.

[The effect of oxygen on the (32)P-labelling of polyphosphates and organic phosphates in Ankistrodesmus braunii in the light]. / Zur Wirkung von Sauerstoff auf die (32)P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht.

Short time incorporation of (32)P was carried out with synchronised algae (young cells) depleted of phosphate. For the separation and determination of the acid-insoluble phosphate fractions of the cells an improved fractionation procedure was applied. In order to exclude competition by carbon dioxide all experiments were done in the absence of CO2.Compared with nitrogen, CO2-free air produces an increase in the labelling of phosphorylated compounds in the light. In strong white light, at high pH, air effects a remarkable increase of (32)P in the acid-insoluble phosphate (P u), mainly in inorganic polyphosphates (P ul), whereas the total phosphate uptake remains almost unchanged. The increase in labelling of acid-insoluble phosphate is, therefore, accompanied by a substantial decrease in the labelling of acid-soluble compounds (P l). In weak white light or in far-red light, at low pH even in strong white or red light, an increase of phosphate uptake and an increased labelling of the acid-stable organic acid-soluble fraction (P os) is observed instead. The effect of oxygen increases somewhat with increasing light intensity up to light saturation, and it increases markedly with increasing oxygen concentration.An essential contribution by oxidative phosphorylation to this oxygen effect can be ruled out on account of its much higher sensitivity to oxygen. Pseudocyclic photophosphorylation is also not regarded as the main force because of its higher oxygen affinity. Occurrence of photorespiration has not been clearly established so far in related algae (Chlorella), and its use for phosphorylation is unknown. A better, although not complete explanation is given by comparing the oxygen effect with the well-known inhibition of photosynthesis by oxygen (Warburg effect), which leads to an increase in glycolate formation and a simultaneous decrease in the pool sizes of carbon reduction cycle intermediates, even in the absence of CO2. Since the photophosphorylation process, as well as the photosynthetic electron flow, seem unaffected by high oxygen concentrations whereas the formation of organic phosphate compounds is partially inhibited, excess ATP may be available for polyphosphate synthesis. This explanation would be consistent with the assumption that polyphosphate-ADP kinase mediates an equilibrium between ATP and polyphosphates, mainly at higher pH. At low pH and in other cases the excess ATP might be available for an increased phosphate uptake and for phosphorylation of endogenous carbohydrates.