It is well established that nitrite reductase (NIR; EC 1.7.7.1) a key enzyme of nitrate reduction - is "induced" by nitrate and light. In the present study with the spinach (Spinacia oleracea L.) seedling the dependency of NIR appearance on nitrate, light and a 'plastidic factor' was investigated to establish the nature of the coaction between these controlling factors. A cDNA clone coding for spinach NIR was available as a probe. The major results we have obtained are the following: (i) The light effect on the appearance of NIR activity occurs through phytochrome. No specific bluelight effect is involved, (ii) Immunotitration data indicate that light affects the appearance of NIR by inducing the de-novo synthesis of the NIR protein, (iii) A multiplicative relationship exists between the action of nitrate and light on NIR appearance. This indicates that the actions of light and nitrate are indeed independent of each other but that both factors operate on the same causal sequence, (iv) Anion-exchange chromatography revealed only a single form of NIR in spinach. Experiments involving plastid photooxidation indicate that this NIR is exclusively plastidic. (v) Northern blot analysis of NIR mRNA showed a strong increase of the steady-state level in the presence of nitrate whereas light had no effect; NIR mRNA was almost undetectable when the plastids were damaged by photooxidation. It is concluded that NIR gene expression in spinach requires positive control by a 'plastidic factor'. Moreover, nitrate exerts a coarse control at the mRNA level whereas fine tuning of NIR protein synthesis is post-transcriptional and is exerted by light, operating via phytochrome.

Nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) are the key enzymes of nitrate reduction. It is well established that the appearance of these enzymes is "induced" by nitrate, and it is generally believed that NR is cytosolic while NIR is plastidic. In mustard (Sinapis alba L.) cotyledons we observed two isoforms of NIR (NIR1 and NIR2) using a chromato-focusing technique. Only one of them (NIR2) disappeared when the plastids were damaged by photooxidation in the presence of Norflurazon. It is concluded that NIR2 is plastidic while NIR1 is extraplastidic and not affected by photooxidation of the plastids. Both isoforms appear to have the same molecular weight (60 kilodaltons, kDa). Two distinct translation products which could be immunoprecipitated with NIR antiserum produced against total NIR from mustard were observed which differed slightly in molecular weight (60 versus 63 kDa). The 63-kDa polypeptide was considered to be the precursor of NIR2. While synthesis of NIR protein depended largely on nitrate, the levels of in-vitro-translatable NIR mRNAs were found to be either independent of nitrate and light (NIR1) or controlled by phytochrome only (NIR2). It appears that phytochrome strongly stimulates the level of mRNA while significant enzyme synthesis (NIR2) takes place only in the presence of relatively large amounts of nitrate. Since an increased enzyme level was strictly correlated with an increase of immunoresponsive NIR protein it is improbable that activation of a precursor plays a role. Rather, it is concluded that, in situ, nitrate controls translation.

We compared the response of NO 3 (-) -induced nitrate-reductase (NR) and nitrite-reductase (NIR) levels in virtually carotenoid-free far-red-light-grown mustard (Sinapis alba L.) cotyledons following a photooxidative treatment of the plastids. The cytosolic localization of NR and the plastidic localization of NIR were confirmed with this approach. Emphasis was on a plastidic factor previously postulated to be involved obligatorily in the transcriptional control of nuclear genes coding for proteins destined for the chloroplast. Photooxidative damage of the plastid would be to destroy the ability of the organelle to send off this signal. Dependency of NIR and NR induction by NO 3 (-) on the plastidic factor is described in detail, and it is concluded that requirement for the plastidic factor is relatively high in the case of NR while factor requirement to allow induction is low in the case of NIR. The data indicate that in the case of NIR the photooxidative damage done to the plastid also affects accumulation of the enzyme directly. Since this effect is absent in the case of cytosolic NR, induction of NR is a particularly suitable system for further molecular studies of the plastidic factor and its mode of action.

Application of nitrate leads to an induction of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of dark-grown mustard (Sinapis alba L.) seedlings, and this induction can strongly be promoted by a far-red-light pretreatment - operating through phytochrome - prior to nitrate application. This light treatment is almost ineffective - as far as enzyme appearance is concerned - if no nitrate is given. When nitrate is applied, the stored light signal potentiates the appearance of NR and NIR in darkness, even in the absence of active phytochrome, to the same extent as continuous far-red light. This action of previously stored light signal lasts for approx. 12 h.Storage of the light signal was measured for NR and NIR. The process shows enzyme-specific differences. Storage occurs in the absence as well as in the presence of nitrate, i.e. irrespective of whether or not enzyme synthesis takes place. The kinetics of signal transduction and signal storage indicate that the formation and action of the stored signal are a bypass to the process of direct signal transduction. Signal storage is possibly a means of enabling the plant to maintain the appropriate levels of NR and NIR during the dark period of the natural light/dark cycle.

It was inferred from previous findings that a plastid-derived factor (plastidic factor) is involved in the transcriptional control of nuclear genes coding for proteins destined for the chloroplast. Photooxidative damage to the plastid destroys the ability of the organelle to give off this factor. Cytosolic enzyme levels are not impaired if plastids are damaged, and morphogenesis of seedlings is normal. The only exception found so far is nitrate reductase, a cytosolic enzyme, which is regulated by the cellas if it were a plastidic protein. In the present study we have shown that the plastids in the mesophyll of mustard (Sinapis alba L.) cotyledons, damaged by 3 h photooxidation in red light (6.8 W·m(-2)) and then returned to darkness or to continuous, non-photooxidative far-red light (cFR), recover from photooxidative damage. The rate of recovery is stimulated by phytochrome (operationally, cFR). Since the cytosolic enzyme nitrate reductase is affected by the different treatments in principally the same way as the levels of plastidic enzymes, we conclude that it is recovery of the plastids' ability to give off the plastidic factor rather than structural recovery which leads to recovery of gene expression and protein (and chlorophyll) re-accumulation. The extent of recovery varied according to the enzyme and this variation could be explained by different plastidic-factor requirements for gene expression. This explanation was confirmed by measurements of translatable mRNAs. It was found that LHCP-gene expression (light-harvesting chlorophyll a/b-binding protein of photosystem II) is far more sensitive to photooxidative damage of the plastids than SSU-gene expression (small subunit of ribulose-1.5-bisphosphate carboxylase). Correspondingly, recovery is expressed to a much greater extent in the latter than in the former case.