Mustard Cotyledons Research Articles

Temporal Pattern of Gene Expression in Cotyledons of Mustard (Sinapis alba L.) Seedlings

AbstractThe time course of appearance of competence to phytochrome (Pfr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light‐mediated accumulation of mRNAs encoding for SSU, CAB and the 23 kDa protein of the oxygen evolving complex of photosystem II (OEC). For each gene family a specific starting point of Pfr‐induced mRNA accumulation was observed (SSU: 42 h; CAB: 36 h; OEC: 30 h). An increase of SSU‐mRNA levels can be detected 24 h after sowing in dark‐grown seedlings whereas for OEC the time points for the increase of mRNA are the same whether the seedlings are kept in darkness or induced by light via Pfr. For all gene families a responsiveness to Pfr (coupling point) could be demonstrated before the starting points. The coupling points are also gene specific (SSU: ca. 12 h; CAB and 23 kDa peptide of OEC: ca. 24 h). The responsiveness to light before the starting point indicates that the light‐induced signal must be stored.

Appearance of nitrite-reductase mRNA in mustard seedling cotyledons is regulated by 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.

Photooxidative damage to plastids affects the abundance of nitrate-reductase mRNA in mustard cotyledons

It was found previously that in the mustard (Sinapis alba L.) seedling (Schuster et al. 1989, Planta 177, 74-83) the action of nitrate and phytochrome on the appearance of cytosolic nitrate reductase (NR) is abolished if the plastids are damaged by photooxidation. In the present study this finding has been corroborated by the following results: (i) the appearance and disappearance of NR activity are strictly correlated with the appearance and disappearance of immunoresponsive NR protein; (ii) the appearance of NR correlates with the appearance of translatable NR mRNA; (iii) photodestruction of the plastids strongly reduces the level of NR mRNA. It is concluded that the dependence of the NR level on the state of the plastids can be detected at the level of its mRNA and is not attributable to an inactivation of the enzyme.

Factors controlling nitrate and ammonium accumulation in mustard (Sinapis alba) seedlings

Prolonged application of ammonium as a source of nitrogen leads to serious physiological and morphological disorders in many plants, including mustard (Sinapis alba L,) seedlings (ammonium toxicity syndrome). Ammonium tolerance was previously observed in mustard seedlings in the presence of considerable amounts of nitrate in the medium. In the present study, the question was addressed as to what extent accumulation of nitrate and ammonium occurs in the mustard seedling and how this relates to ammonium toxicity and tolerance. Emphasis was on light control of accumulation in the attached cotyledons. Both NQ3 and NH4 became strongly accumulated in the mustard cotyledons once the concentration in the medium exceeded 1 mM, In the cotyledons, we measured concentrations > 30 mM in the case of nitrate and > 50 mM in the case of ammonium 4 days after sowing. Accumulation of inorganic nitrogen in the mustard cotyledons did not depend on photosynthesis nor on intact chloroplasts. However, the rate of nitrate accumulation was strongly stimulated by light, operating through phytochrome, while ammonium accumulation was not affected by light in short‐term experiments, i,e, within 24 h and only weakly (and probably indirectly) in long‐term light.We conclude that strong interaction between NQ3 and NHJ is characteristic for accumulation of inorganic nitrogen. In the presence of NQ3, accumulation of NH3 is diminished and, to a lesser extent, vice versa. Using [15N]‐labelling it was found that incorporation of inorganic nitrogen into organic compounds was stimulated strongly whenever nitrate and ammonium were provided simultaneously. It appears from the data that accumulation of nitrate is strictly controlled by the mustard cotyledons while the accumulation of ammonium is not. The data indicate that the level of ammonium is governed primarily by the rate of ammonium assimilation. Stimulation of ammonium assimilation by simultaneously applied nitrate appears to explain nitrate‐mediated ammonium tolerance in the mustard plant.

The participation of cis-4-cyclohexene-1,2-dicarboximide in the inhibition of light-controlled growth responses

cis-4-Cyclohexene-1,2-dicarboximide (CHDC) and several structural analogues inhibit the germination of light-sensitive seeds but not that of non-photoblastic seeds. Other cell elongation processes mediated by red light, viz. fern spore germination, primary leaf unrolling in barley and enlargement of bean leaf discs are also inhibited by CHDC, in a manner which is reversible by benzyladenine (BA) or Ca 2+ ions. Light-inhibited elongation is not reversed by CHDC. The inhibition of growth is not convened to light-mediated systems, however, for growth of lettuce hypocotyls in darkness, and in light on gibberellin, is prevented by CHDC, as is radish hypocotyl growth. Auxin-induced growth of pea stem segments is not inhibited, on the other hand. Other events not related to cell elongation, such as β-amylase and anthocyanin production in mustard cotyledons, are inhibited by CHDC. Thus CHDC, while inhibitory to light-controlled cell elongation will also inhibit other light-mediated processes.

Characterization of β-amylase from Sinapis alba cotyledons

β-Amylase from mustard ( Sinapis alba) cotyledons was purified to homogeneity by affinity chromatography on a starch column. M r determination by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration revealed that the native enzyme is a monomer of 58 000. The analysis of products of enzyme action on amylose by paper chromatography and optical rotation revealed the exclusive formation of β-maltose as a product confirming the molecular nature of the purified enzyme as β-amylase. The substrate specificity of mustard β-amylase was akin to other plant β-amylases. The K m value for β-amylase with amylose as substrate was 0.24%. The enzyme was fairly stable at or below ambient temperature, to repeated freezing and thawing and exposure to a wide pH range (3–8). The inclusion of dithiothreitol in storage buffer improved the stability of enzyme. The enzyme was susceptible to denaturation (70–90% inactivation) by heavy metal ions (Cu 2+, Pb 2+, Ag +) and sulphydryl reagents such as p-chloromercuribenzoate, indicating the sulphydryl nature of the enzyme. In contrast, α-cyclodextrin, a competitive inhibitor of β-amylase caused only a mild reduction in enzyme activity.

The Effect of Light and Exogenously Supplied Ammonium Ions on Glutamate Dehydrogenase Activity and Isoforms in Young Mustard (Sinapis alba L.) Seedlings

AbstractGlutamate dehydrogenase (GDH, E.C. 1.4.1.3) of mustard cotyledons was investigated during the first 4 days of seedling development. The enzyme was found to be composed of seven catalytically active isoforms (each with a molecular mass of 270 kDa) which exhibited a charge heterogeneity when investigated by isoelectric focusing. Antibodies against the purified isoform 7, raised in rabbits, cross‐reacted with each of the isoforms in Western blotting experiments. In addition, each of the isoforms was composed of four immunopositive reacting polypeptides with 19, 21, 23 and 25 kDa. During development of the seedlings, a shift in the isoform pattern towards the more acidic forms was found which was more pronounced when the seedlings were supplied with 15 mM NH4Cl. The time course of changes in total GDH level can be correlated with the time course of disappearance of storage proteins. Both parameters are negatively regulated by light possibly via the photoreceptor, phytochrome. There are some indications that GDH in young mustard cotyledons mainly acts in the deaminating direction.

AN ANALYSIS OF PHYTOCHROME‐MEDIATED THRESHOLD CONTROL OF HYPOCOTYL GROWTH IN MUSTARD (Sinapis alba L.) SEEDLINGS†

Abstract—Hypocotyl elongation in mustard (Sinapis alba L.) seedlings is known to be controlled by phytochrome (Pfr) through a threshold response. This phytochrome‐mediated threshold response was studied in detail with the following results: (i) The Pfr threshold value required to suppress hypocotyl growth is much lower (0.03% Prr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%) than those threshold valued observed previously in threshold control by hook phytochrome of appearance of ‘potential capacity for photophosphorylation’ and lipoxygenase appearance in the mustard cotyledons (1.25% Ptr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%). This probably explains why hypocotyl elongation is so extremely sensitive to light, (ii) The Pfr threshold value controlling hypocotyl growth is a system constant, independent of total phytochrome content, developmental age and actual growth rate, (iii) Threshold control of hypocotyl elongation is unaffected by the removal of the cotyledons and half of the hook. However, removal of the whole hook totally eliminates any light control over the residual hypocotyl growth, (iv) After termination of the threshold control, the hypocotyl growth rate immediately returns to precisely that found in untreated dark control even though the partial growth rates of the different parts of the hypocotyl are quite different, relative to their dark controls. Obviously, the organ grows as an integrated unit.It is concluded that the all‐or‐none threshold control over hypocotyl growth is exerted from the plumular hook. It appears that the hook can send off phytochrome all‐or‐none signals in both directions, to the cotyledons and to the hypocotyl.

Regulation of the appearance of glutamine synthetase in mustard (Sinapis alba L.) cotyledons by light, nitrate and ammonium

During transformation of mustard seedlings cotyledons from storage organs to photosynthetically competent leaves, a process which occurs during the first 4 d after sowing, total glutamine-synthetase (GS, EC 6.3.1.2) activity increases from zero to the high level usually observed in green leaves. In the present study we have used ion-exchange chromatography to separate possible isoforms of GS during the development of the cotyledons. The approach failed since we could only detect a single form of GS, presumably plastidic GS, under all circumstances tested. The technique of selective photooxidative destruction of plastids in situ was applied to solve the problem of GS localization. It was inferred from the data that the GS as detected by ion-exchange chromatography is plastidic GS.The regulatory role, if any, of light, nitrate and ammonium in the process of the appearance of GS in the developing cotyledons was investigated. The results show that nitrate and ammonium play only minor roles. Light, operating via phytochrome, is the major regulatory factor.

β-Amylase from Mustard (Sinapis alba L.) Cotyledons

A polyclonal antiserum against mustard (Sinapis alba L.) beta-amylase was obtained by injecting a homogeneously purified enzyme preparation in rabbits. The formation of beta-amylase specific antibodies was confirmed by staining the precipitin line in double diffusion gel for beta-amylase activity. The monospecificity of antiserum against mustard beta-amylase was also ascertained by Western blotting. The antiserum efficiently recognised both the denatured and the native form of beta-amylase, but it did not cross-react with other higher plant beta-amylase. The mode of photoregulation of beta-amylase activity in mustard cotyledons was investigated by a variety of immunochemical techniques. Immunotitration experiments ruled out the possible contribution of enzyme activation/inactivation in photoregulation of beta-amylase activity. The use of single radial immunodiffusion, rocket immunoelectrophoresis, and immunotitration confirmed that the light mediated increase in beta-amylase activity quantitatively corresponds with the increase in beta-amylase protein level. The in vivo labeling with l-[(35)S] methionine and pulse chase studies of in vivo labeled beta-amylase protein revealed that the photoregulated increase in beta-amylase activity in mustard cotyledon exclusively results from an increase in the rate of de novo synthesis of beta-amylase protein against a very low background rate of enzyme degradation.

Differential regulation by phytochrome of the appearance of plastidic and cytoplasmatic isoforms of glutathione reductase in mustard (Sinapis alba L.) cotyledons

An increase of glutathione reductase (GR; EC 1.6.4.2) activity during the transformation of mustard (Sinapis alba L.) cotyledons from storage organs to photosynthetically competent leaves was previously found to be controlled by light acting via phytochrome (Drumm, H., Mohr, H., Z. Naturforsch. 28c 559-563, 1973). Two isoforms of GR could be separated by disc electrophoresis. In the present study we have applied ionexchange chromatography to separate isoforms of GR during the development of the cotyledons. Furthermore, the technique of in situ photooxidation of plastids was used to distinguish between plastidic and cytoplasmatic isoforms. The isoform GR2 is the plastidic enzyme, as shown by its sensitivity to photooxidative treatment, while GR1 is a cytoplasmatic protein not affected by photooxidative treatment of plastids. Both isoforms are promoted by phytochrome but with different time courses. The appearance of GR1 is independent of the integrity of the plastids, as one might expect. However, unexpectedly, the phytochrome-mediated re-appearance of GR2 after a photooxidative treatment is much less affected by photooxidative destruction of the plastids, i.e. by the lack of a particular "plastidic factor", than was to be expected from previous experience with typical plastidic proteins. An interpretation of this finding must await measurements at the level of GR2 mRNA.

Effect of nitrate, ammonium, light and a plastidic factor on the appearance of multiple forms of nitrate reductase in mustard (Sinapis alba L.) cotyledons

In mustard (Sinapis alba L.) cotyledons, four different forms of nitrate reductase (NR) can be separated by anion-exchange chromatography. Two of these forms (NR1 and NR2) appear in the presence of NO 3 (-) while the other two (NR3 and NR4) appear as a response to the application of NH 4 (+) as the sole nitrogen source. In the presence of NH4NO3, NR3 appears to be superimposed on nitrate-induced NR1 and NR2 while the NH 4 (+) -induced appearance of NR4 is totally abolished in the presence of equimolar amounts of NO 3 (-) . The appearance of NR1, NR2 and NR3 is strongly stimulated by red light pulses which operate via the far-red-absorbing form of phytochrome (Pfr), whereas the appearance of NR4 requires continuous light (likewise operating through pytochrome). Continuous red light is more effective in this case than continuous far-red light. Analysis of the data shows that the mode of action of phytochrome (Pfr) is the same in the case of the appearances of NR1 and NR2, whereas it is quantitatively different in the case of NR3 and totally different in that of NR4. A 'plastidic factor' has previously been postulated to be obligatorily involved in the transcriptional control of nuclear genes encoding for proteins destined for the chloroplast. Photooxidative damage of the plastid is postulated to destroy the ability of the organelle to produce this signal. If the plastids are damaged by photooxidation, the action of nitrate and phytochrome on the appearance of NR is abolished. The plant cell regulates the appearance of nitrate-induced NR, which is cytosolic, as if it were a plastidic protein. The appearance of NR3 depends on the plastidic factor in principally the same way as that of NR1 and NR2 whereas NR4 is totally independent of the plastidic factor. The data document particular kinds of interaction between controlling factors (light, nitrate, ammonium, plastidic factor) which affect gene expression in plants. These intricacies of regulation have so far not been considered in molecular studies on NR-gene expression.

Phytochrome control of the accumulation and rate of synthesis of ascorbate oxidase in mustard cotyledons

Mustard seedlings were grown in darkness or exposed to far red light. Investigations into the enzyme ascorbate oxidase (AO) in extracts from cotyledons exposed to 36 h far red light and from dark controls revealed: (1) the activity of AO increases up to 7-fold upon light treatment. (2) The amount of AO decreases after 36 h light treatment compared to dark controls; this has been measured by ELISA using a horseradish peroxidase conjugate employing either a colorimetric or an enhanced chemiluminescent assay; the latter was applied both to samples in microtitre plates and on nitrocellulose filters. (3) The rate of AO synthesis, as measured by incorporation of [ 35S]methionine into AO, appears to be lower in extracts of cotyledons exposed to 36 h light than in dark controls; this has been monitored by employing nitrocellulose filters coated with AO-specific antibodies followed by autoradiography. The results are inconsistent with a hypothesis that far red light leads to an increase in the rate of synthesis of this enzyme; rather they suggest post-translational control of AO activity by phytochrome.

Time courses for phytochrome-induced enzyme levels in phenylpropanoid metabolism (phenylalanine ammonia-lyase, naringenin-chalcone synthase) compared with time courses for phytochrome-mediated end-product accumulation (anthocyanin, quercetin)

Time course for changes in the levels of enzymes characteristic of general phenylpropanoid metabolism (phenylalanine ammonia-lyase, PAL; EC 4.3.1.5) and of the flavonoid-glycoside branch pathway (naringenin-chalcone synthase, CHS; EC 2.3.1.74) were measured in the cotyledons of mustard (Sinapis alba L.) seedlings and compared with the rates of accumulation of related end products (anthocyanin and quercetin). Induction of enzyme levels and of end-product accumulation was carried out with red and far-red (FR) light, operating via phytochrome. The data are compatible with the concept that the phytochrome-mediated appearance of enzymes such as PAL and CHS is indeed a prerequisite for the appearance of anthocyanins and flavonols. However, there is no close correlation between enzyme levels and the rates of synthesis of end products which could justify the identification of specific rate-limiting enzymes. Rather, the data indicate that there is a second phytochrome-dependent step, beyond enzyme induction, where the actual rate of flavonoid accumulation is determined. Anthocyanin and quercetin accumulation respond differently to light. However, the relative action of continuous FR, red light pulses and 'stored phytochrome signal' is the same in both cases. This indicates that the mode of operation of phytochrome is the same in both cases. The two syntheses differ only in the degree of responsiveness towards phytochrome. The time course for changes in CHS levels in continuous FR, i.e. under conditions of phytochrome photosteady state, is similar to the time course for PAL levels whereas the time courses in darkness, following transfer from FR to darkness, are totally different. In the case of CHS, a transient rise is observed whereas, with PAL, an instantaneous drop in enzyme level occurs after transfer from FR to darkness. It is concluded that the 'stored phytochrome signal' operates in darkness in the case of CHS but not in the case of PAL.

Changes in ribulose‐1,5‐bisphosphate carboxylase and its translatable small subunit mRNA levels during senescence of mustard (Sinapis alba) cotyledons

Senescence of mustard (Sinapis alba L.) cotyledons was studied by monitoring the levels of ribulose‐1,5‐bisphosphate carboxylase (RuBP carboxylase, EC 4.1.1.39) activity, enzyme protein and translatable mRNA of the small subunit (SSU‐mRNA) of RuBP carboxylase. In continuous white light (cWL) RuBP carboxylase began to decline 6 days after sowing and the decrease in SSU‐mRNA preceded the drop in enzyme protein. The decline in RuBP carboxylase was more pronounced when the seedlings were transferred to darkness. This dark‐mediated senescence was affected by phytochrome (Pfr) primarily by retardation of enzyme degradation and to a lesser extent by the maintenance of gene expression. When seedlings were reirradiated with cWL or red light after being kept for 6–8 days in darkness the levels of enzyme protein and SSU‐mRNA again increased. Thus, even after long periods of darkness the seedlings retained their ability to respond to light with renewed gene expression which was affected by Pfr. Under short‐day conditions (8 h light/16 h dark), almost no decline of RuBP carboxylase occurred, whereas SSU‐mRNA decrease followed the same pattern as in cWL or darkness.

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

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.

Control by phytochrome of the appearance of ribulose-1,5-bisphosphate carboxylase and the mRNA for its small subunit

We have measured levels of ribulose-1,5-bisphosphate carboxylase (RuBPCase) and levels of in-vitro-translatable mRNA for the small subunit (SSU) of RuBPCase up to 96 h after sowing in mustard (Sinapis alba L.) cotyledons, in order to investigate to what extent the rate of enzyme synthesis is related to the level of SSU-mRNA. Both enzyme and mRNA level are controlled strongly by phytochrome, but the rate of RuBPCase accumulation was found to be unrelated to the level of translatable SSU-mRNA. As an example, it was found that the amount of SSU-mRNA in far-red light (FR)-grown mustard seedlings doubles between 54 and 84 h after sowing while the rate of RuBPCase accumulation remains constant over this period. Since the holoenzyme shows zero turnover during this period it is concluded that the rate of enzyme synthesis remains constant although the level of SSU-mRNA increases strongly. Following an FR→dark transition, with different levels of physiologically active phytochrome (Pfr) established at the end of the light period, no correlation was found between the time course of mRNA levels in darkness and the rate of enzyme synthesis. Rather, the data indicate that there is at least one translational or post-translational regulatory step which is also phytochrome-dependent. It is concluded that coarse control of the appearance of translatable SSU-mRNA is essential for RuBPCase to appear at a high rate but that fine tuning by phytochrome of the actual appearance of RuBPCase is not transcriptional.

RAPID TRANSMISSION OF A PHYTOCHROME SIGNAL FROM HYPOCOTYL HOOK TO COTYLEDONS IN MUSTARD (Sinapis alba L.)

Abstract— In mustard (Sinapis alba L.) cotyledons appearance of ‘potential capacity’ to phosphorylate is controlled by phytochrome (Pfr) through a threshold, all‐or‐none reaction. Phytochrome operates only if the hypocotyl hook is connected to the cotyledons for at least 2.5 min after the end of a 1 min inductive light pulse. Partial irradiations of hook and cotyledons show that in the case of induction of ‘potential capacity’ rapid transmission of the Pfr signal occurs from the hypocotyl hook to the cotyledons. Phytochrome within the cotyledons is totally ineffective in this regard. A 3 min red light pulse suffices for full transmission of the signal. It seems that an explanation of the facts we have observed requires a revision of the present views about communication between the organs of a plant.

Sustainment of Uninterrupted Increase of β-amylase Activity in Mustard (Sinapis alba L.) Cotyledons during Light/Dark Cycles

Summary In the cotyledons of mustard (Sinapis alba L.) seedlings, phytochrome-mediated increase in the rate of de novo synthesis of β-amylase (E.C. 3.2.1.2) under continuous red light involves a stable regulatory element (≪transmitter≫) in the signal transduction chain which has a life time of about 12 h and acts in an allor -none fashion. Therefore continuous red light can be quantitatively substituted by alternate cycles of red light and darkness, each of 12 h duration. However, when phytochrome is reverted to the Pr form before the onset of the daily dark cycle, seedlings require a 17 h light/7 h dark period to sustain uninterrupted β-amylase synthesis as in continuous red light. The same result is obtained with a 6 h red light/6 h dark period. Using inhibitors of transcription and translation it is established that the transmitter is a stable β-amylase mRNA. The uninterrupted increase in β-amylase level in light/dark cycles results from the continued presence of stable β-amylase mRNA in the dark periods and the resumption of β-amylase mRNA synthesis at the onset of the next light period. These results indicate that under natural conditions β-amylase accumulation in mustard cotyledons would proceed normally through the daily night period.

Physiological characterization of a plastidic signal required for nitrate-induced appearance of nitrate and nitrite reductases

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.