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.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

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     

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     

Regulation of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from pea seedlings: rapid posttranslational phytochrome-mediated decrease in activity and in vivo regulation by isoprenoid products.

Brief red light irradiation (5 min) of etiolated pea seedlings causes a 40 to 50% decline in microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and far red reversal experiments indicate phytochrome mediation. The response is apparent at the earliest assay time, 5 min after irradiation, hence there is little or no lag period; a substantial change occurs within 10 min, and a 24% decrease at 1 h. Activity remains low for about 24 h. The response half-time is about 25 min. Cordycepin affects activity only after 3 h; cycloheximide inhibits only 6% at 1 h and has no effect on activity for at least 20 to 30 min after it blocks protein synthesis. It is concluded that phytochrome regulates reductase activity indirectly through a posttranslational mechanism which causes a stable change in enzyme activity; there is no indication that phytochrome acts by binding directly to the reductase. The decline in reductase activity following irradiation, or cycloheximide treatment, does not follow first-order kinetics. Mixing experiments suggest increased levels of a reductase inactivator in irradiated tissues. The low reductase activity in green seedlings is increased by treatment with dibutyryl-cyclicAMP. Abscisic acid and cholesterol applied to etiolated seedlings reduce activity of the enzyme but gibberellic acid has no effect. However, abscisic acid and cholesterol added to reaction mixtures do not inhibit activity. The metabolic consequences of the rapid light-induced enzyme response may trigger, or contribute to, later biochemical responses previously assumed to be under more direct phytochrome control.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Molecular biology, genetics and regulation of nitrite reduction in higher plants

Nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.6.6.1) carries out the six-electron reduction of nitrite to ammonium ions in the chloroplasts/plastids of higher plants. The complete or partial nucleotide sequences of a number of nitrite reductase apoprotein genes or cDNAs have been determined. Deduced amino acid sequence comparisons have identified conserved regions, one of which probably is involved in binding the sirohaem/4Fe4S centre and another in binding the electron donor, reduced ferredoxin. The nitrite reductase apoprotein is encoded by the nuclear DNA and is synthesised as a precursor carrying an N-terminal extension, the transit peptide, which acts to target the protein to, and within, the chloroplast/plastid. In those plants examined the number of nitrite reductase apoprotein genes per haploid genome ranges from one (barley, spinach) to four ( Nicotiana tabacum ). Mutants defective in the nitrite reductase apoprotein gene have been isolated in barley. During plastidogenesis in etiolated plants, synthesis of nitrite reductase is regulated by nitrate, light (phytochrome), and an uncharacterised 'plastidic factor' produced by functional chloroplasts. In leaves of green, white-light-grown plants up-regulation of nitrite reductase synthesis is achieved via nitrate and light and down-regulation by a nitrogenous end-product of nitrate assimilation, perhaps glutamine. A role for phytochrome has not been demonstrated in green, light-grown plants. Light regulation of nitrite reductase genes is related more closely to that of photosynthetic genes than to the nitrate reductase gene. In roots of green, white-light-grown plants nitrate alone is able to bring about synthesis of nitrite reductase, suggesting that the root may possess a mechanism that compensates for the light requirement seen in the leaf.     

Photophysiology of turion germination in Spirodela polyrhiza (L.) Schleiden. X. Role of nitrate in the phytochrome-mediated response

It was shown previously that light-dependent germination of turions of Spirodela polyrhiza (Lemnaceae) is mediated by the photoreceptor phytochrome [Appenroth & Augsten (1990) Photochemistry and Photobiology 52, 61–65]. In the present study, we found that this photoresponse depends on nitrate in the surrounding medium both during after-ripening (under natural conditions occurring in winter) and during germination after light-induction (in spring). The action of nitrate in the germination response is neither related to the induction of nitrate reductase nor to the rate of uptake of ¹⁵NO₃^?. Moreover, two-factor analysis (phytochrome, nitrate) revealed a multiplicative coaction, i.e. independent action of both factors in mediation of germination. The notion that nitrate is a nutritional prerequisite in phytochrome-mediated germination of turions, is supported by the following facts: (1) Nitrate-requirement during germination was strongly increased by nitrate starvation during after-ripening prior to germination. (2) Ammonium could substitute for nitrate. (3) Nitrate uptake by the turions was unaffected by phytochrome and very pronounced even at low concentrations (0.07 mol m^?3) in the medium. With regard to the phytochrome-induced chain of events, it is concluded that nitrate is a prerequisite during a specific developmental phase. Nitrate is not a regulatory element within the chain. In an ecological sense, however, nitrate contents of the aquatic system regulate the germination of turions.     

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, nitrate movement, and induction of nitrate reductase in etiolated pea terminal buds

The role of phytochrome in the induction of nitrate reductase of etiolated field peas (Pisum arvense L.) was examined. Terminal bud nitrate concentration increased in darkness, and the increase correlated with induction of nitrate reductase following brief exposure of intact plants to red, blue, far red, and white lights. Brief light exposure of intact plants stimulated nitrate uptake and induction of nitrate reductase by terminal buds subsequently excised and incubated on nitrate solution in darkness; exposure of excised buds in contact with nitrate led to less uptake but more induction. Nitrate and nitrate reductase activity both declined during incubation with water, irrespective of light treatment. Nitrate enrichment of intact terminal buds and uptake into excised buds and increases in nitrate reductase activity were all red/far red reversible. Dimethyl sulfoxide (1%, v/v) and sugars (sucrose 0.5%, glucose 1, w/v), although stimulating nitrate uptake into excised tissue in darkness, failed to enhance nitrate reductase activity over dark controls. Phytochrome may regulate nitrate reductase via both nitrate movement and a general mechanism such as enhancement of protein synthesis.     

Time-dependent changes in the responsiveness to light of phytochrome-mediated anthocyanin synthesis

Abstract. It is well established that seedlings of mustard ( Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far-red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag- phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.     

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.     

Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare cv. Midas)

Abstract San 9789 (norflurazone) blocks carotenoid synthesis which allows chlorophyll bleaching in the light, and has been used recently as a tool to study phytochrome responses without interference from photosynthetic pigments. By using this herbicide, we have found that nitrate reductase activity and light dependent nitrite reduction were lost simultaneously from achlorophyllous areas of barley leaves, with the green areas of the leaf tip still showing high activities. By contrast nitrate reductase is still present in the roots of herbicide treated plants. We suggest that intact chloroplasts are required for the presence of nitrate reductase in barley leaves.     

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 Interactive Effects of Phytochrome, Nitrate and Thiourea on the Germination Response to Alternating Temperatures in Seeds of Ranunculus sceleratus L.: A Quantal Approach

Probert, R. J., Gajjar, K. H. and Haslam, I. K. 1987. The interactiveeffects of phytochrome, nitrate and thiourea on the germinationresponse to alternating temperatures in seeds of Ranunculussceleratus L.: A quantal approach.—J. exp. Bot. 38: 1012–1025. The interactive effects of phytochrome, potassium nitrate andthiourea on the germination response to alternating temperaturesin achenes (seeds) of Ranunculus sceleratus L. were studied.Using thermogradient bars, high levels of germination were recordedover a broad range of alternating temperatures providing seedsreceived daily irradiations. Reduced germination in temperaturecycles with a relatively long warm phase was related to thelevel of the active form of phytochrome (Pfr). Dose-responseexperiments to red light (R) and temperature shifts showed thatthe actions of Pfr and alternating temperatures were interdependent.Maximum germination was recorded when intermittent pulses ofR were combined with daily 4 h temperature shifts from 16°Cto 26°C. Whilst probit analysis showed that potassium nitrateand thiourea both increased population sensitivity to temperatureshifts, thiourea was a more potent stimulant. Although the effectof both chemicals was dependent on phytochrome photo-equilibriumthe threshold level of Pfr required for thiourea action wasclearly much lower than that required for nitrate action. Thioureapotentiated a response to daily temperature shifts even whenPfr was at a low, normally inhibitory level. These results indicatedifferent mechanisms of action for potassium nitrate and thioureain relation to phytochrome controlled seed germination. Key words: Phytochrome, nitrate, thiourea, alternating temperatures, germination