Phytochrome regulation of nitrate reductase in wheat

In excised wheat leaves, the activity of nitrate reductase was enhanced by a brief pulse of red light and this increase was reversed by far-red light irradiation. Even under continuous far-red light, nitrate reductase activity increased by 258% after 18 h. When leaves were kept in distilled water during exposure to red light and then transferred to potassium nitrate, there was no difference in endogenous nitrate concentration. The nitrate reductase activity was the same whether leaves were floated in potassium nitrate or in distilled water during irradiation. Partial to complete inhibition of enzyme activity was observed when leaves were incubated in actinomycin-D and cycloheximide respectively, following 4 h of red light irradiation.In vitro irradiation of extract had no significant effect on nitrate reductase activity     

Rapid activation by phytochrome of nitrate reductase in the cotyledons of Sinapis alba

Summary Nitrate reductase in the cotyledons of etiolated seedlings of Sinapis alba L. responds rapidly to the addition of nitrate. The response is inhibited by cycloheximide at low concentrations. The enzyme is also under phytochrome control. Five minutes of red light irradiation leads instantaneously to a 45% increase in enzyme activity. Increases in activity, linear with respect to time and with no lag phases are promoted by continuous far-red or blue irradiation. These increases are insensitive to cycloheximide. Thus, light and nitrate act through different mechanisms in controlling nitrate reductase activity and phytochrome does not act via controlling the rate of synthesis of the enzyme.Abbreviation cot pr pair of cotyledons     

Temporal separation of two components of phytochrome action

Abstract In germinating seedlings of Sinapis alba nitrate reductase activity as assayed in vivo becomes accessible to phytochrome control between 15 and 17 h after sowing. Phytochrome operates via the high irradiance reaction to control nitrate reductase activity in the period 15 to 20 h after sowing. Both continuous red light and far-red light elicit this response with a strong fluence rate dependency being apparent in each case. The induction of nitrate reductase activity by light pulses at 20 h after sowing is greatly influenced by red light pre-treatments (operating through phytochrome) given between 0 and 15 h after sowing. Low fluence rate pre-treatments reduce the effectiveness of a subsequent pulse to below the level of a dark control whilst high fluence rate pre-treatments greatly increase the effectiveness of a subsequent pulse.     

Signal storage in phytochrome action on nitrate-mediated induction of nitrate and nitrite reductases in mustard seedling cotyledons

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.Abbreviations cD continuous darkness - cFR continuous far-red light - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light - RG9-light long wavelength far-red light obtained with RG9 glass filter - - Ptot total phytochrome (Pr+Pfr) Professor Wilhelm Nultsch mit guten Wünschen zum 60. Geburtstag     

Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons

In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.     

Phytochrome Regulation of Nitrite Reductase--a Chloroplast Enzyme--in Etiolated Maize Leaves

Nitrite reductase in the excised etiolated leaves of maize showedthe photoreversibility by red and far-red light. Five minutesof red light illumination lead to a 130% increase in the enzymeactivity which was reversed by far-red light. The kinetics ofnitrite reductase activity under continuous far-red light showeda lag phase of 1 hr. (Received January 17, 1981; Accepted February 20, 1981)     

Influence of Age and Light on the Distribution and Development of Nitrate Reductase in Greening Barley Leaves

The relation between leaf age and the induction of nitrate reductase activity by continuous and intermittent light was studied with barley seedlings (Hordeum vulgare L. cv. Club Mariout). In general, nitrate reductase activity declined as the period of growth in darkness was extended beyond 5 days. Maximum activity was found near the leaf tip while activity was lowest in the morphologically youngest tissue near the base of the lamina. Increased activity was observed after continuous illumination of dark-grown seedlings for 24 hours. The increase in activity in response to light was greatly reduced when the dark pretreatment period was extended beyond 8 days. The amount of nitrate reductase activity present in the different sections of the leaf was closely related to the amount of polyribosomes present. The pattern of chlorophyll accumulation closely parallelled that of increases in nitrate reductase activity. The initial lag in the induction of nitrate reductase activity was removed by a 10-minute light treatment 6 hours before placing dark-grown barley seedlings in light. The enzyme was also induced under flashing light with various dark intervals. These induction curves closely resembled those of chlorophyll accumulation under the same conditions. The development of photosynthetic CO₂ fixation follows the same induction pattern in this system. Our results suggest that photosynthetic products may be required for the induction of significant levels of nitrate reductase activity in leaves of dark-grown seedlings, although other light effects may not be discounted.     

Circadian expression of the light-harvesting protein of Photosystem II in etiolated bean leaves following a single red light pulse: Coordination with the capacity of the plant to form chlorophyll and the thylakoid-bound protease

The appearance of the light harvesting II (LHC II) protein in etiolated bean leaves, as monitored by immunodetection in LDS-solubilized leaf protein extracts, is under phytochrome control. A single red light pulse induces accumulation of the protein, in leaves kept in the dark thereafter, which follows circadian oscillations similar to those earlier found for Lhcb mRNA (Tavladoraki et al. (1989) Plant Physiol 90: 665–672). These oscillations are closely followed by oscillations in the capacity of the leaf to form Chlorophyll (Chl) in the light, suggesting that the synthesis of the LHC II protein and its chromophore are in close coordination. Experiments with levulinic acid showed that PChl(ide) resynthesis does not affect the LHC II level nor its oscillations, but new Chl a synthesis affects LHC II stabilization in thylakoids, implicating a proteolytic mechanism. A proteolytic activity against exogenously added LHC II was detected in thylakoids of etiolated bean leaves, which was enhanced by the light pulse. The activity, also under phytochrome control, was found to follow circadian oscillations in verse to those in the stabilization of LHC II protein in thylakoids. Such a proteolytic mechanism therefore, may account for the circadian changes observed in LHC II protein level, being implicated in pigment-protein complex assembly/stabilization during thylakoid biogenesis.Abbreviations Chl chlorophyll - CL continuous light - D dark - FR far-red light - LA levulinic acid - LHC II light-harvesting complex serving Photosystem II - PChl(ide) protochlorophyllide - PCR protochlorophyllide oxidoreductase - R red light     

STIMULATION OF NITRATE NET UPTAKE AND REDUCTION BY RED AND BLUE LIGHT AND REVERSION BY FAR-RED LIGHT IN THE GREEN ALGA ULVA RIGIDA1

The steady-state levels of nitrate, nitrite, and ammonium were estimated in the green alga Ulva rigida C. Agardh in darkness after addition of 0.5 mM KNO₃ and irradiation with red (R) and blue (B) light pulses of different duration (5 and 30 min). The net uptake of nitrate was very rapid. Seventy-five percent of the nitrate added was consumed after 60 min in darkness. Although uptake was stable after R or B, efflux of nitrate occurred within 3 h in the dark control and when R or B were followed by far-red (FR) irradiation. The internal nitrate concentration after 3 h in darkness was similar after R and B light pulses; however, the intracellular ammonium was higher after R than after B. The intracellular nitrate and ammonium decreased when FR tight pulses were applied immediately after R or B. Thus, the involvement of phytochrome in the transport of nitrate and ammonium is proposed. Nitrate reductase activity, measured by the in situ method, was increased by both R and B light pulses. The effect was partially reversed by FR light. Nitrate reductase activity was higher after 5 min of R light than after 5 min of B. However, after 30-min light pulses, the relative increase in activity was reversed for R and B. We propose that phytochrome and a blue-light photoreceptor are involved in regulation of nitrogen metabolism. Nitrate uptake and reduction correlates with previously detected light-regulated accumulation of protein in Ulva rigida under the same experimental conditions.     

Effects of red, far red, and blue light on enhancement of nitrate reductase activity and on nitrate uptake in etiolated rice seedlings

The effects of red (R), far red (FR), or blue light (B) on the enhancement of nitrate reductase (NR) activity and on nitrate uptake in etiolated rice seedlings were examined. On 5-minute illumination followed by 12-hour dark, R caused marked increase of NR activity, but FR and B caused only slight increase. Illumination with 560 ergs per square centimeter per second of R for 5 minutes caused maximal increase. The effect of R was almost completely counteracted by subsequent illumination with 2,000 ergs per square centimeter per second of FR for 10 minutes, indicating that NR induction was mediated by phytochrome. Exogenous supply of inducer nitrate was not required during the 5-minute illumination and the R-FR cycles, if the seedlings were transferred to nitrate solution at the beginning of the dark incubation. NR activity in the shoots was found high when shoots were illuminated but was low when only roots were illuminated. On continuous illumination for 12 hours, B had more effect on NR increase than R.     

Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.     

Photocontrol of seed germination in Impatiens wallerana Hook. f.

General characteristics of light sensitivity of Impatients wallerana seeds were investigated. Germination was absolutely dependent on light, irrespective of temperature. High percentages of germination were obtained by exposure to long periods of illumination or, alternatively, to several repeated short irradiations with red light. In this case, responsiveness to light was not altered by increasing either the initial incubation period in darkness or the dark intervals between short exposures. Effects of red light were reversed by far-red light, thus demonstrating the involvement of phytochrome. Evidence was presented for an interactive effect, of unknown physiological nature between red and far-red light on the germination of the seeds.Abbreviations P_r phytochrome, red light absorbing form - Pfr phytochrome far-red absorbing form     

Development of cytosol and chloroplast aldolases during germination of spinach seeds

The total activity of aldolase (EC 4.1.2.13) and the activities of cytosol and chloroplast aldolase were determined in seeds, cotyledons, primary leaves and secondary leaves of spinach (Spinacia oleracea L., cv. Monopa) during germination. Total aldolase activity in cotyledons increased from low levels to a low maximum in the dark after one week and to a high maximum in white light after three to four weeks and declined thereafter. The activity in primary and secondary leaves started to rise strongly from the 18th and 26th days, respectively, up to the 42nd day of germination. The levels of aldolase activity paralleled the development of leaf area, chlorophyll content and protein content per leaf except that the leaf area of cotyledons continued to increase steadily up to the 42nd day after the maximum of aldolase activity was reached. Resolution of cytosol- and chloroplast-specific isoenzymes by chromatography on diethylaminoethylcellulose indicated that in the light the cytosol enzyme represented approx. 8% of the total activity in cotyledons, primary and secondary leaves throughout germination, and the chloroplast enzyme represented the remaining 92%. Only in cotyledons of dark-grown seedlings was the cytosol aldolase between 25 and 50% of the total activity. Seeds contained almost exclusively a cytosol aldolase. In cotyledons the increase of total activity in the light was specifically the consequence of an increase in chloroplast aldolase while the cytosol aldolase was little affected by light. The light effect was mediated by phytochrome as demonstrated by classical induction and reversion experiments with red and far-red light and by continuous far-red light treatment.Abbreviation DEAE-cellulose diethylaminoethylcellulose     

Studies on Greening of Etiolated Seedlings II. Leaf Greening by Phytochrome Action in the Embryonic Axis

We could demonstrate that greening of primary bean leaves in etiolated seedlings of Phaseolus vulgaris cv. Limburg can be controlled by a selective light-pretreatment of the embryonic axis. This light-induced interorgan synergism proved to be a phytochrome-mediated process. The red/farred photoreversible effect on the embryonic axis seems to be primarily linked to changes in the energy metabolism of the primary leaves. Phototransformation of the protochlorophyll present and pigment synthesis are very dependent upon an adequate supply of biochemical energy. When the embryonic axis is selectively pre-exposed to red light for a short time, respiration is markedly enhanced in the leaves and photosynthesis starts immediately upon illumination of the etiolated leaves after an incubation period of optimal length in the dark. The stimulatory effect of the red pretreatment on leaf respiration and photosynthetic capacity could be abolished to the level of the dark controls by a subsequent far-red irradiation on the embryonic axis. It is therefore postulated that phytochrome plays a regulatory role in interorgan cooperation. The metabolic changes involved in photomorphogenesis of etiolated seedlings are closely related to changes in energy production. Our data indicate that the primary act of phytochrome becomes operative at the biochemical level by its directional influence on the energy balance of the cell and coordinates the use of metabolic energy within a tissue and between organs.     

Phytochrome involvement in the induction of resistance to dark abscission by malformin

The active portion of the visible spectrum which is required for malformin to produce leaves which are resistant to dark abscission from cuttings of Phaseolus aureus is red light. Abscission resistance was partially to almost completely lost by far irradiation prior to dark incubation. Although Ethrel, an ethylene releasing compound, stimulated dark abscission of resistant and control leaves, resistance was not lost because control leaves always abscised at a greater rate. The participation of phytochrome in the induction of abscission resistance by malformin is indicated.Abbreviations Pfr far-red absorbing form of the phytochrome system - R red radiation - FR far-red radiation - D dark     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Influence of cytokinins and phytochrome on nitrate reductase activity in etiolated leaves of maize

Of the different hormones tested, cytokinins stimulated nitrate-induced nitrate reductase (NR) activity in the dark. The optimal stimulation was obtained at 16 hr and this was sensitive to tungstate, 6-methylpurine and cycloheximide. The cytokinin stimulation of NR activity was further enhanced by brief irradiation with red light, but this effect was not noticed when leaves were exposed to far-red light. Both kinetin and red light, when given together, or given with a darkness interruption, stimulated the NR activity more than with either of them alone.     

Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba L.) seedling as affected by nitrate,phytochrome and photooxidative damage of plastids

Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard (Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that intact plastids are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some plastidic signal is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle.Abbreviations c continuous - D darkness - FR far-red light - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.1.13) - NF Norflurazon - NIR nitrite reductase (EC 1.7.7.1.) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red light obtained with RG9 glass filter - R red light - RG9-light long wavelenght far-red light obtained with RG9 glass filter - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - WL white light - WL_s strong white light (28 W m^-2)     

The Effect of Age on the Phytochrome-Mediated Chlorophyll Formation in Dark-grown Bean Leaves

The effect of red and far-red treatment on chlorophyll synthesis in dark-grown bean leaves was studied at various ages. Although the effect was pronounced in the old leaves, no effect was observed in the young ones (4 days old). In the 5-day old leaves a measurable effect of red light pretreatment can be observed, whereas the far-red reversal effect was not observed. — The length of the dark period between the red pretreatment and the continuous illumination is also age dependent. Leaves older than 6 days show a maximum at about six hours, while in the young leaves the red light effect increases with the time of dark incubation up to the 24 hours tested. — The reversal effect of far-red light on protochlorophyllide regeneration was also examined. The far-red light has no reversal effect on leaves younger than 6 days old, while on the old leaves it has such an effect.