Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling the appearance of ferredoxin-dependent glutamate synthase in the Scots pine (Pinus sylvestris L.) seedling

The appearance of NADH- and ferredoxin (Fd)-dependent glutamate synthases (GOGATs) was investigated in the major organs (roots, hypocotyl and cotyledonary whorl) of the Scots pine seedling. It was found that cytosolic NADH-GOGAT (EC 1.4.1.14) dropped to a low level during the experimental period (from 4 to 12 d after sowing) and was not significantly affected by light. On the other hand, plastidic Fd-GOGAT (EC 1.4.7.1) increased strongly in response to light. Whereas similar amounts of NADH-GOGAT were found in the different organs, Fd-GOGAT was mainly found in the cotyledons even in the presence of nitrate. Protein chromatography revealed only a single Fd-GOGAT peak. No isoforms were detected. Experiments to investigate regulation of the appearance of Fd-GOGAT in the cotyledonary whorl yielded the following results: (i) In darkness, neither nitrate (15 mM KNO₃) nor ammonium (15 mM NH₄Cl) had an effect on the appearance of Fd-GOGAT. In the light, nitrate stimulated Fd-GOGAT activity by 30% whereas ammonium had no effect. The major controlling factor is light. (ii) The action of long-term white light (100 W · m^–2) could be replaced quantitatively by blue light (B, 10 W · m^–2). Since the action of long-term far-red light was very weak, operation of the High Irradiance Reaction of phytochrome is excluded. On the other hand, light-pulse experiments with dark-grown seedlings showed the involvement of phytochrome. (iii) Red light, operating via phytochrome, could fully replace B, but only up to 10 d after sowing. Thereafter, there was an absolute requirement for B for a further increase in the enzyme level. It appears that the operation of phytochrome was replaced by the operation of cryptochrome (B/UV-A photoreceptor). (iv) However, dichromatic experiments (simultaneous treatment of the seedlings with two light beams to vary the level of the far-red-absorbing form of phytochrome (Pfr) in blue light) showed that B does not affect enzyme appearance if the Pfr level is low. It is concluded that B is required to maintain responsiveness of Fd-GOGAT synthesis to phytochrome (Pfr) beyond 10 d after sowing.Abbreviations and Symbols B blue light - c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light - HIR high-irradiance reaction of phytochrome - NADH-GOGAT nicotinamide-dinucleotide-dependent glutamate synthase (EC 1.4.1.14) - R red light - RG9 long-wavelength far-red light defined by the properties of the Schott glass filter (_RG9<0.01) - Pfr/Ptot far-red-absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 und Schwerpunkt Physiologie der Bäume). We thank E. Fernbach for his help with the dichromatic experiments.     

Treatment with natural human interferon alpha of a CML-patient with antibodies to recombinant interferon alpha-2b

A patient with Philadelphia-chromosome positive chronic myelogenous leukemia developed interferon antibodies on treatment with recombinant interferon alpha-2b. Clinically this event corresponded with progressive disease. No cross-reactivity of antibodies with human leukocyte interferon was found by Western blot. Treatment was switched to human leukocyte interferon with an obvious clinical effect: WBC was reduced and platelet count stabilized, but the effect was transient and no hematologic remission was achieved. Human leukocyte interferon may be an alternative in CML-patients with neutralizing antibodies to recombinant interferon alpha.     

Carotenoid composition in milo (Sorghum vulgare) shoots as affected by phytochrome and chlorophyll

The composition of coloured carotenoids in the milo shoot was investigated quantitatively (high performance liquid chromatography) during light-mediated plastidogenesis, including the time span of photodelay as caused by medium and high light fluxes. It was found that as long as only the far-red-absorbing form of phytochrome operates, the carotenoid pattern remains virtually the same as in complete darkness (violaxanthin and lutein as major constituents, traces of -carotene). On the other hand, the pattern changes dramatically in white or red light with increasing amounts of chlorophyll (lutein and -carotene dominate, -carotene showing the strongest relative increase). Photodelay during the early phase of plastidogenesis affects the carotenoid composition strongly. Increase of neoxanthin, violaxanthin and -carotene contents are diminished while lutein accumulation proves resistant towards chlorophyll-mediated photoinhibition. The photodelay can be diminished by an appropriate light pretreatment. The data indicate that light-mediated control over carotenoid accumulation is exerted at three levels: i) a coarse control through phytochrome, ii) fine tuning in connection with chlorophyll accumulation, iii) stabilization of holocomplexes against photodecomposition.Abbreviations GG14 high fluence rate green-yellow light - HPLC high-performance liquid chromatography - Chl chlorophyll - WL_w weak white light (1200 lx) - WL_m medium flux white light (12000 lx)     

Phytochrom-induzierte Enzymbildung (Phenylalanindesaminase), ein schnell ablaufender Prozeß

Zusammenfassung Bei Erstbelichtung des Senfkeimlings mit Dauer-Dunkelrot tritt der P₇₃₀-abhängige Anstieg der Phenylalanindesaminase-Aktivität erst mit einer lag-Phase von 1,5 Std ein (Abb. 3, 4 unten). Bei einer Zweitbelichtung (Programm: Erstbelichtung — längere Dunkelperiode — Zweitbelichtung) fehlt die lag-Phase (Abb. 4, oben). Die Enzymaktivität steigt sofort linear an. Da der Anstieg der Enzymaktivität wahrscheinlich auf eine de novo Synthese von RNS und Enzymprotein zurückzuführen ist (Tabelle), so erscheint der Schluß berechtigt, daß P₇₃₀ sehr rasch eine differentielle Genaktivierung mit anschließender Enzymsynthese bewirken kann, falls die Gene der Aktivierung durch P₇₃₀ zugänglich sind. Die relativ lange lag-Phase nach Einsetzen der Erstbelichtung benötigt das P₇₃₀ offenbar dazu, die potentiell aktiven Gene (P₇₃₀) für das P₇₃₀ zugänglich zu machen. Das Problem der primären lag-Phase ist in einer vorangegangenen Arbeit zur P₇₃₀-abhängigen Anthocyansynthese ausführlich diskutiert worden (vgl. Lange, Bienger und Mohr, 1967).

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Phytochrome-mediated enzyme formation (Phenylalanine deaminase) as a rapid process

Summary In previous papers we have reported (Mohr and Durst, 1966a, b) that synthesis of phenylalanine deaminase (EC 4.3.1.5), an important enzyme of phenolic metabolism, can be stimulated by the physiologically active phytochrome (=P₇₃₀) in the mustard seedling. The data of the present paper suggest that induction of this enzyme is a rapid process if the gene in question is easily accessible for the activating action of P₇₃₀.The seedlings were irradiated with continuous standard far-red light. Longtime irradiation with far-red will maintain a low but virtually constant level of P₇₃₀ in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of P₇₃₀ will virtually cease. — Fig. 3 and Fig. 4, lower part, show the kinetics of enzyme induction by P₇₃₀ in an etiolated seedling. The initial (or primary) lag-phase after the onset of far-red is 1.5 hours. If, however, a seedling which has been pre-irradiated with 12 hours of far-red is kept in darkness for 6 hours and is then re-irradiated with far-red no lag-phase for the action of the second irradiation can be found. Enzyme activity increases immediately after the onset of far-red. Since the action of the second irradiation as measured by increase of enzyme activity can be inhibited by relatively low doses of Puromycin and Cycloheximide (table) we conclude that the re-appearance of P₇₃₀ leads to de novo synthesis of enzyme protein. — Application of Actinomycin D (10 g/ml) only partially inhibits the action of the second irradiation as measured by increase of enzyme activity. This finding was to be expected. In preceding papers (e.g. Mohr and Bienger, 1967) it has been concluded that genes which have once been activated by P₇₃₀ remain less sensitive towards Actinomycin D even when P₇₃₀ has disappeared. Taking into account all available data the conclusion seems to be justified that the induction of enzyme synthesis by P₇₃₀ (i.e. differential gene activation followed by enzyme synthesis) is a rapid process if the genes are accessible for the action of P₇₃₀. The relatively long initial lag-phase (1.5 hours) is needed to make the potentially active genes (P₇₃₀) accessible for the action of P₇₃₀. The problem of how the initial lag-phase can be understood has been dealt with more in detail in a previous paper on phytochrome-mediated anthocyanin synthesis (Lange, Bienger and Mohr, 1967).

     

Protein- und RNS-Gehalt des Hypokotyls beim stationären Wachstum im Dunkeln und unter dem Einfluß von Phytochrom (Keimlinge von Sinapis alba L.)

Zusammenfassung Das Wachstum des Hypokotyls wurde an Restkeimlingen ohne Kotyledonen (Abb. 1) untersucht. Die Wachstumsgeschwindigkeit ist in dem von uns untersuchten Zeitraum sowohl im Dunkeln als auch unter dem Einfluß von P₇₃₀ (Dauer-Dunkelrot) praktisch konstant. Obgleich sich die Wachstumsgeschwindigkeiten im Dunkeln und im Dauer-Dunkelrot um den Faktor 4 unterscheiden, hat das Dunkelrot keinen signifikanten Einfluß auf den Gesamt-Proteingehalt des Hypokotyls (bzw. der durchschnittlichen Hypokotylzelle). Der Proteingehalt nimmt im Dunkeln und im Licht kontinuierlich ab. Auch der Gesamt-RNS-Gehalt zeigt innerhalb des Versuchszeitraums eine Abnahme, die unter dem Einfluß von Dunkelrot früher einsetzt als im Dunkeln. — Man kann aus den Daten der vorliegenden Arbeit schließen, daß nur ein kleiner Teil des Gesamt-Proteins und der Gesamt-RNS einer Zelle mit dem Zellwachstum unmittelbar in Verbindung gebracht werden kann.

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Protein and RNA contents of the hypocotyl during steady state growth lengthening in the dark and under the influence of phytochrome (seedlings of sinapis alba L.)

Summary Inhibition of hypocotyl lengthening by phytochrome can be regarded as a prototype of a negative photoresponse. The hypothesis has been advanced (Schopfer, 1967) that negative photoresponses are the consequence of a differential gene repression which is exerted by P₇₃₀, the active phytochrome. This hypothesis is mainly based on experiments with specific inhibitors of RNA- and protein synthesis. —The present paper is part of an experimental program which has been designed to check this hypothesis.—Continuous irradiation with standard far-red has been used to establish a virtually stationary concentration of P₇₃₀ over the whole period of experimentation (36–60 hours after sowing). To correlate more strictly the growth response of the hypocotyl with molecular changes in this organ the axis system without cotyledons has been used (Fig. 1). Even under these conditions the growth rate of the hypocotyl is nearly constant in light (continuous far-red) and dark during the whole period of experimentation (36–60 hours after sowing) (Fig. 2, 3). It is known from earlier experiments that cell division in the hypocotyl are very rare during this period and that there is virtually no increase in the DNA contents of the organ during the period of our experimentation (Weidner, 1967). Obviously the number of cells per hypocotyl is virtually constant between 36 and 60 hours after sowing. Organ (i.e. hypocotyl) lengthening is nearly exclusively due to cellular lengthening.—If we follow the protein contents of the hypocotyl we find (Fig. 4) that the total protein of the organ decreases steadily in spite of the fact that the organ grows at a constant rate. There is no significant difference in protein contents between dark-grown and far-red grown systems although the growth rates differ by a factor of 4 (Fig. 2, 3).—The situation is some-what different with respect to total RNA (Fig. 5). The RNA contents eventually decrease in far-red as well as in dark-grown systems but the decrease is significantly faster in the far-red treated systems than in the dark controls.—It is concluded that only a very small part of the total RNA and total protein of a cell can be related to the control of cellular growth. Changes in bulk RNA and bulk protein obviously do not necessarily reflect changes in the growth rate or growth capacity of an organ or a cell.