Relationship between nitrate reduction and proteolytic activity

stored for 96 h in liquid nitrogen without significant loss of activity. Addition of FAD to crude . Although the relationship between nitrate reduction and NADH oxidation is buffer B plus 3%. BSA, or buffer B plus a protease inhibitor mix recom-. Assimilatory nitrate reductase (NR) of higher plants is a most interesting state also independently affect the activity or induction of the NR protease(s). The ratio of these three NR‐forms is variable, depending on external conditions. Nitrate Reductase and Protease Activities during Grain provide some insight into the relation between newly reduced and remobil-.

In the transgenic N. When these plants were irrigated with a high concentration mM of KNO3 for about 1 week, nitrite accumulated during the night, but disappeared again after about 1 h of high light intensity in the morning. This disappearance of nitrite in the light is linked with the onset of photosynthesis, which generates reduced ferredoxin used by nitrite reductase, and therefore stimulates further metabolism of nitrite.

Chlorosis of young leaves developed in the S mutant after about 3 weeks. The importance of the regulatory serine for the avoidance of nitrite accumulation is clearly demonstrated when cut roots or leaves of N. Only roots from the transgenic S continue to excrete nitrite during the 5 h incubation time Fig. In leaves this was even more clear; nitrite excretion was negligible after 1 h in all lines tested except for S for which nitrite excretion continued at a high rate for at least 5 h Lea et al.

A special enzyme, nitric oxide synthase, is responsible for NO formation in mammals. NO is known to be mutagenic in Salmonella and assumed to be mutagenic in mammals and leads to the production of peroxynitrite Wink et al.

The accumulation of nitrite may, therefore, not only result in cytotoxic effects, but also influence growth and development. NO emission from leaves and roots of N. NR is not the only source of NO synthesis in plants. It has been shown very recently that Arabidopsis possesses a nitric oxide synthase gene, which was discovered through its homology to a snail enzyme, implicated in NO production Guo et al. Before that, another plant NO synthase gene was identified as a variant of the P protein of the glycine decarboxylase complex Chandok et al.

The question which remains open is, is the NO produced by NR actually biologically active and if not, is there any mechanism which could possibly allow the plants to discriminate between the different sources of NO production?

However, they are largely hydrophilic and all have a region with acidic amino acids Nussaume et al. Since this region shows great variation among species it is unlikely to be essential for the function of the enzyme, but is likely to have a role in regulation. This suggests that different mechanisms could be involved in NR inactivation in the dark as opposed to illuminated plants exposed to low CO2 concentrations where there is an excess of reducing power but less sugars.

Indeed, in the former, NR is clearly inactivated by phosphorylation of the Ser residue since, when this residue is mutated, NR activity is no longer inhibited. It is known that several kinases phosphorylate NR and these could have specific roles for the inactivation of NR in response to different factors such as the absence of reducing power and sugars. For instance the plants Snf1 homologues, of which the third NR kinase peak is a member, may respond more specifically to variations in sugar levels.

Diurnal variations Nitrate reductase expression varies during the day and night Lillo et al.

The highest activity of NR is generally observed during the first part of the photoperiod, NR activity then declines during the latter part of the photoperiod and dark period. Diurnal variations of NR activity are still clearly pronounced in the plants C1 although the gene is constitutively transcribed US Lea etal. For example, in spinach, three different kinases phosphorylate NR which leads to the inactivation of the enzyme.

Calcium fluxes in the cell may, therefore, result in changes in NR kinase activity and thereby influence the phosphorylation state of NR. Cations are also important in the activation of the already phosphorylated NR.

It is probable that an interacting site for divalent cations is also present on the NR enzyme itself. This probable redox control by reduced molecules NADH or thiol compounds could provide a link between nitrate reduction and photosynthesis.

For the inactivation of R. Phosphorylation and importance for degradation of NR Protease activity, inactivating or degrading NR in vitro has been frequently reported, but the exact way that initiation of NR degradation takes place in vivo is still unknown Callis, ; MacKintosh and Meek, Attempts to test for the possible involvement of ubiquitination and the proteasome in the degradation of Arabidopsis and spinach NR were negative MacKintosh and Meek, In prolonged darkness or when nitrate is withdrawn from plants, NR protein and activity decline in plants.

Light is not a direct signal to activate NR. Even in continuous strong light, NR becomes inactive when CO2 is absent. Thus, photosynthesis is required for NR activation.

Most probably, assimilates exported out of the chloroplast function as signals. Indeed, NR can be activated in the dark by feeding sugars to the leaves. Moreover, dark inactivation of NR in a starchless mutant of N. All of these responses indicate that NR activation state is sensitive to metabolites and may be explained by the observation that in vitro, the NR protein kinase is inhibited by physiological concentrations of hexose monophosphates Kaiser et al.

It makes nitrate reduction sensitive to stomatal resistance. Thus, when plants close stomata under drought in order to preserve water, not only photosynthesis drops but NR becomes less active. Response of leaf NR activation to nitrate supply Variations in nitrate supply usually do not change the activation state of NR.

Only in rare cases where NR contents were extremely low due to persistent nitrate deficiency Man et al. Again, the reason for that is not yet known. Normally, in tobacco plants with high nitrate supply, synthesis of NR mRNA in leaves may start in the late night phase to reach a maximum around midday, whereas NR protein activity increases in the first half of the day and remains constant or decreases slightly from midday until darkness.

Interestingly, when nitrate supply to barley plants stopped and the internal nitrate concentrations became extremely low, NR protein and activity decreased very strongly during the day, indicating rapid NR degradation Man et al.

In that situation NR was still activated in the dark, whereas normally NR degradation e. Modulation of NR in roots: Assimilate supply from the shoot may vary only slowly within hours or days, as does the nitrate supply. However, there is one natural situation where root NR is also rapidly modulated: It has long been known that NR is more active in anoxic plant tissues than in air, but the reason for this is still unclear.

It has been shown that under sudden anoxia, in darkened leaves, but also in roots, NR was activated within minutes Glaab and Kaiser, ; Botrel et al. It is known that the cytosol of anoxic cells is acidified from the normal pH 7. However, other explanations may seem possible. During anoxia, the reduction of externally added nitrite is very low or absent Botrel et al.

But together with the activation of NR, anoxia always leads to a strong accumulation of nitrite and to nitrite excretion by the roots.

Surprisingly, in anoxic leaf tissues, the reduction of nitrate and the accumulation of nitrite are still much lower than should be expected from the high NR activity determined in the extracts. Again, that indicates that in roots, as in leaves, nitrate reduction rates are not only dictated by the NR activation state, but also by other factors such as substrate NADH? Whether the high nitrite production by anoxic tissues fulfils a physiological purpose, is not yet known.

Production of the weak acid nitrite and excretion of undissociated nitric acid may help to stabilize cytosolic pH. However, preliminary measurements of cytosolic pH gave no clear evidence for that. Alternatively, nitrate reduction may decrease the rate of alcoholic or lactic acid fermentation.

Lactic acid especially may become toxic, as it is not as easily excreted as alcohol or nitric acid. As in leaves, sugar feeding also activates NR in normoxic roots Botrel and Kaiser, It was concluded that assimilate translocation from shoot to root will also affect the nitrate reduction rate of roots, but this is probably a slow response.

In leaves, NR activity decreased whereas in roots in increased with increasing salinity. Partly, this was due to changes in leaf nitrate contents. This latter maximum disappeared with salinity, and accordingly the increase in NR protein and activity in the early light phase was slowed down.

Thus, the existing amount of NR protein depends not only on the rate of synthesis, but also on the rate of degradation. NR synthesis is high in the light and low in the dark Weiner and Kaiser, Indeed, NR protein in darkened leaves appeared more stable whenever it was activated by any of these treatments for review see Kaiser et al. However, these conclusions may be questioned according to recent data Cotelle et al.