Ilse Bienger

Eine neue Beweisführung für die Hypothese einer differentiellen Genaktivierung durch Phytochrom 730

SummaryPhytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) can be regarded as a prototype of a “positive” photoresponse. “Positive” photoresponses are those which are characterized by an initiation or an increase of biosynthetic or growth processes. The initial lag-phase of this type of photoresponse is relatively long. In the case of anthocyanin synthesis of the mustard seedling it takes about 3 hours after the initial formation of P730, the active phytochrome, until anthocyanin synthesis can be measured (Fig.). This period of time has been called initial (or primary) lag-phase.In earlier papers (e.g. Lange and Mohr, 1965; Mohr, 1966a, b; Schopfer, 1967) we have advanced and supported the hypothesis that the positive photoresponses (in the above sense) of the mustard seedling can be explained by a differential gene activation through P730. Those genes of which the activity can be started or increased by P730 have been called potentially active genes (P730).In order to verify this hypothesis further we have been trying to characterize the processes which occur during the initial lag-phase after the onset of continuous far-red light. There is good evidence (Hartmann, 1966; Clarkson and Hillman, 1967) that under continuous far-red a low but virtually stationary concentration of the active P730 can be maintained in the seedlings tissues over a period of many hours. Consequently we are dealing with steady state conditions of P730 when we investigate the process of anthocyanin accumulation under continuous far-red.In the present paper we present data which indicate that P730 exerts two functions during the initial lag-phase. First, it eliminates a barrier before the potentially active genes insolfar as it makes these genes accessible for the activating action of P730. Secondly, P730 starts the activity of potentially active genes. To maintain gene activity the continuous presence of P730 is required. On the other hand, it a gene has once been “opened” to the action of P730 it remains easily accessible for the activating action of P730 even in the case when P730 disappeared and gene activity ceased for an extended period of time. After the re-appearance of P730 the gene can be activated almost immediately. These conclusions have been derived from the following facts.1.The initial lag-phase after the onset of far-red is always in the order of 3 hours (Fig.). If, however, a seedling which was preirradiated with 12 hours of far-red is kept in darkness for an extended period and is then re-irradiated with far-red no lag-phase for the action of the second irradiation can be found even when anthocyanin synthesis had already ceased during the preceding dark period (Fig.).ZusammenfassungNach Erstbelichtung des Senfkeimlings mit Dauer-Dunkelrot zeigt die Anthocyansynthese, eine typische “positive Photomorphose”, eine mehrere Stunden währende lag-Phase. Bei einer Zweitbelichtung (Programm: Erstbelichtung — längere Dunkelperiode — Zweitbelichtung) fehlt die lag-Phase (Abb.). — Auch die Zweitbelichtung dürfte die Anthocyansynthese über eine Genaktivierung, die eine Enzymsynthese zur Folge hat, auslösen (Tabelle). — Die wichtigsten Konsequenzen: P730, das aktive Phytochrom, kann die Enzymsynthese sehr rasch bewirken, falls die Gene der Aktivierung durch P730 zugänglich sind. Die lange lag-Phase nach Einsetzen der Erstbelichtung benötigt das P730 offenbar dazu, die potentiell aktiven Gene (P730) für das P730 zugänglich zu machen. Die diesbezüglichen Änderungen, vermutlich in der Umgebung der Gene, bleiben auch dann erhalten, wenn P730 verschwindet. P730 scheint also zwei distinkte Funktionen zu haben, die man experimentell trennen kann: Es macht die Gene zugänglich und es aktiviert die betreffenden Gene.

Die Steigerung der durch Phytochrom bewirkten Anthocyansynthese des Senfkeimlings (Sinapis alba L.) durch Chloramphenicol

SummaryChloramphenicol (CAP) within a certain range of concentration (about 20–30μg/ml) increases the rate of far-red mediated anthocyanin accumulation in the mustard seedling (Sinapis alba L.) (Fig. 1). The lag-phase after the onset of far-red and the time of termination of anthocyanin synthesis are not influenced by the presence of the antibiotic (Fig. 2). With and without CAP we observe a constant rate of anthocyanin accumulation over a period of at least 24 hours after the lag-phase at all far-red intensities investigated (Fig. 2). The percentage increase of the rate of anthocyanin accumulation which is due to CAP (20 μg/ml) is independent of the far-red intensity applied and always amounts to about 25% (Table). Formally one can conclude that CAP and far-red seem to act as two independent factors in a multiplicative system. On the “molecular” level the observations can possibly be explained as follows: CAP at a concentration of 20 μg/ml inhibits protein synthesis of the plastids. This inhibition leads to an increase of the pool of phenylalanine in the cotyledons. Because the small concentration of CAP does not interfere with protein synthesis (enzyme synthesis) in the cytoplasm, far-red mediated anthocyanin synthesis can proceed normally. Since phenylalanine acts as a precursor of flavonoids the increased pool of this substance will lead to an increase in the rate of anthocyanin accumulation.ZusammenfassungChloramphenicol (CAP) steigert in einem gewissen Konzentrationsbereich (etwa 20–40 μg/ml) die durch Dunkelrot (DR) ausgelöste Anthocyansynthese des Senfkeimlings. Die lag-Phase sowie der Zeitpunkt der Beendigung der Anthocyansynthese werden durch das Antibioticum nicht beeinflußt. Mit und ohne CAP beobachtet man bei allen untersuchten DR-Intensitäten eine konstante Akkumulationsrate an Anthocyan über einen Zeitraum von mindestens 24 Std. —Die Steigerung der Akkumulationsrate durch CAP (20 μg/ml) liegt stets im Bereich von 25%, auch wenn die DR-Intensität auf die Hälfte oder ein Viertel gesenkt wird (Wirkung des CAP=25% · Wirkung des DR). Man kann daraus formal schließen, daß CAP und DR unabhängig voneinander die Anthocyansynthese beeinflussen. “Molekular” läßt sich der Sachverhalt folgendermaßen deuten: CAP hemmt bei einer Konzentration von 20 μg/ml die Proteinsynthese der Plastiden. Dadurch wird der Phenylalanin-pool in den Kotyledonen erhöht. Da diese geringe CAP-Konzentration die cytoplasmatische Proteinsynthese noch nicht hemmt, kann die Anthocyansynthese ungestört ablaufen. Da Phenylalanin als eine Vorstufe der Flavonoidsynthese fungiert, führt das erhöhte Angebot an dieser Substanz zu einer Steigerung der Anthocyansynthese.

Die Photomodulation der Akkumulationsrate von Ascorbinsäure beim Senfkeimling (Sinapis alba L.) durch Phytochrom

SummaryAscorbic acid accumulation in the mustard seedling is controlled by Pfr(active phytochrome). Kinetic studies demonstrate that Pfrexerts a rapid and fully reversible control over the steady state rate of ascorbic acid accumulation. Following the terminology of Weisz (1967) for this type of metabolic control the term “photomodulation by Pfr” is used.—The control by Pfris independent of RNA synthesis. Therefore regulation of gene activity is probably not involved in photomodulation of the rate of ascorbic acid accumulation.—There is only a limited period within which Pfrcan control ascorbic acid accumulation. This period is fixed by the time pattern of “primary” differentiation in the seedling. There is no interaction between photomodulation by Pfrand control by “primary” differentiation of ascorbic acid accumulation.

Further experiments on the effect of actinomycin D on phytochrome-mediated anthocyanin synthesis

Phytochrome-mediated anthocyanin synthesis of the mustard seedling can be blocked by moderate concentrations of Actinomycin D (10 μg/ml in the solution around the seedling) if the substance is applied before the onset of far-red light (Fig. 1). If, however, the seedlings are irradiated with far-red for 6 hours, transferred to the dark for one hour, incubated with Act. D for 3 hours (in the dark) and then re-irradiated with continuous far-red, anthocyanin synthesis can only partially be inhibited (Fig. 2). - There are good arguments that the physiological action of the small stationary concentration of P730 which is left in the plant after we turn off the far-red light will virtually stop at the moment when the light is turned off (WAGNER and MOHR, 1966a). Furthermore the P730-dependent mRNAs which are involved in anthocyanin synthesis seem to be shortlived (LANGE and MOHR, 1965; MOHR and SENF, 1966; DURST and MOHR, 1966). Considering these arguments and the results of the present paper we cannot but conclude that genes which have once been activated by P730 will not return to the original state-at least with respect to Act. D sensitivity-even when the activating (or de-repressing) agent (P730 in our case) has disappeared.SummaryPhytochrome-mediated anthocyanin synthesis of the mustard seedling can be blocked by moderate concentrations of Actinomycin D (10 μg/ml in the solution around the seedling) if the substance is applied before the onset of far-red light (Fig. 1). If, however, the seedlings are irradiated with far-red for 6 hours, transferred to the dark for one hour, incubated with Act. D for 3 hours (in the dark) and then re-irradiated with continuous far-red, anthocyanin synthesis can only partially be inhibited (Fig. 2). — There are good arguments that the physiological action of the small stationary concentration of P730 which is left in the plant after we turn off the far-red light will virtually stop at the moment when the light is turned off (Wagner and Mohr, 1966a). Furthermore the P730-dependent mRNAs which are involved in anthocyanin synthesis seem to be shortlived (Lange and Mohr, 1965; Mohr and Senf, 1966; Durst and Mohr, 1966). Considering these arguments and the results of the present paper we cannot but conclude that genes which have once been activated by P730 will not return to the original state—at least with respect to Act. D sensitivity—even when the activating (or de-repressing) agent (P730 in our case) has disappeared.

[The increase of phytochrome-mediated anthocyanin synthesis in the mustard seedling (Sinapis alba L.) by chloramphenicol].

SummaryChloramphenicol (CAP) within a certain range of concentration (about 20–30μg/ml) increases the rate of far-red mediated anthocyanin accumulation in the mustard seedling (Sinapis alba L.) (Fig. 1). The lag-phase after the onset of far-red and the time of termination of anthocyanin synthesis are not influenced by the presence of the antibiotic (Fig. 2). With and without CAP we observe a constant rate of anthocyanin accumulation over a period of at least 24 hours after the lag-phase at all far-red intensities investigated (Fig. 2). The percentage increase of the rate of anthocyanin accumulation which is due to CAP (20 μg/ml) is independent of the far-red intensity applied and always amounts to about 25% (Table). Formally one can conclude that CAP and far-red seem to act as two independent factors in a multiplicative system. On the “molecular” level the observations can possibly be explained as follows: CAP at a concentration of 20 μg/ml inhibits protein synthesis of the plastids. This inhibition leads to an increase of the pool of phenylalanine in the cotyledons. Because the small concentration of CAP does not interfere with protein synthesis (enzyme synthesis) in the cytoplasm, far-red mediated anthocyanin synthesis can proceed normally. Since phenylalanine acts as a precursor of flavonoids the increased pool of this substance will lead to an increase in the rate of anthocyanin accumulation.ZusammenfassungChloramphenicol (CAP) steigert in einem gewissen Konzentrationsbereich (etwa 20–40 μg/ml) die durch Dunkelrot (DR) ausgelöste Anthocyansynthese des Senfkeimlings. Die lag-Phase sowie der Zeitpunkt der Beendigung der Anthocyansynthese werden durch das Antibioticum nicht beeinflußt. Mit und ohne CAP beobachtet man bei allen untersuchten DR-Intensitäten eine konstante Akkumulationsrate an Anthocyan über einen Zeitraum von mindestens 24 Std. —Die Steigerung der Akkumulationsrate durch CAP (20 μg/ml) liegt stets im Bereich von 25%, auch wenn die DR-Intensität auf die Hälfte oder ein Viertel gesenkt wird (Wirkung des CAP=25% · Wirkung des DR). Man kann daraus formal schließen, daß CAP und DR unabhängig voneinander die Anthocyansynthese beeinflussen. “Molekular” läßt sich der Sachverhalt folgendermaßen deuten: CAP hemmt bei einer Konzentration von 20 μg/ml die Proteinsynthese der Plastiden. Dadurch wird der Phenylalanin-pool in den Kotyledonen erhöht. Da diese geringe CAP-Konzentration die cytoplasmatische Proteinsynthese noch nicht hemmt, kann die Anthocyansynthese ungestört ablaufen. Da Phenylalanin als eine Vorstufe der Flavonoidsynthese fungiert, führt das erhöhte Angebot an dieser Substanz zu einer Steigerung der Anthocyansynthese.

New evidence in favour of the hypothesis of differential gene activation by phytochrome 730

SummaryPhytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) can be regarded as a prototype of a “positive” photoresponse. “Positive” photoresponses are those which are characterized by an initiation or an increase of biosynthetic or growth processes. The initial lag-phase of this type of photoresponse is relatively long. In the case of anthocyanin synthesis of the mustard seedling it takes about 3 hours after the initial formation of P730, the active phytochrome, until anthocyanin synthesis can be measured (Fig.). This period of time has been called initial (or primary) lag-phase.In earlier papers (e.g. Lange and Mohr, 1965; Mohr, 1966a, b; Schopfer, 1967) we have advanced and supported the hypothesis that the positive photoresponses (in the above sense) of the mustard seedling can be explained by a differential gene activation through P730. Those genes of which the activity can be started or increased by P730 have been called potentially active genes (P730).In order to verify this hypothesis further we have been trying to characterize the processes which occur during the initial lag-phase after the onset of continuous far-red light. There is good evidence (Hartmann, 1966; Clarkson and Hillman, 1967) that under continuous far-red a low but virtually stationary concentration of the active P730 can be maintained in the seedlings tissues over a period of many hours. Consequently we are dealing with steady state conditions of P730 when we investigate the process of anthocyanin accumulation under continuous far-red.In the present paper we present data which indicate that P730 exerts two functions during the initial lag-phase. First, it eliminates a barrier before the potentially active genes insolfar as it makes these genes accessible for the activating action of P730. Secondly, P730 starts the activity of potentially active genes. To maintain gene activity the continuous presence of P730 is required. On the other hand, it a gene has once been “opened” to the action of P730 it remains easily accessible for the activating action of P730 even in the case when P730 disappeared and gene activity ceased for an extended period of time. After the re-appearance of P730 the gene can be activated almost immediately. These conclusions have been derived from the following facts.1.The initial lag-phase after the onset of far-red is always in the order of 3 hours (Fig.). If, however, a seedling which was preirradiated with 12 hours of far-red is kept in darkness for an extended period and is then re-irradiated with far-red no lag-phase for the action of the second irradiation can be found even when anthocyanin synthesis had already ceased during the preceding dark period (Fig.).ZusammenfassungNach Erstbelichtung des Senfkeimlings mit Dauer-Dunkelrot zeigt die Anthocyansynthese, eine typische “positive Photomorphose”, eine mehrere Stunden währende lag-Phase. Bei einer Zweitbelichtung (Programm: Erstbelichtung — längere Dunkelperiode — Zweitbelichtung) fehlt die lag-Phase (Abb.). — Auch die Zweitbelichtung dürfte die Anthocyansynthese über eine Genaktivierung, die eine Enzymsynthese zur Folge hat, auslösen (Tabelle). — Die wichtigsten Konsequenzen: P730, das aktive Phytochrom, kann die Enzymsynthese sehr rasch bewirken, falls die Gene der Aktivierung durch P730 zugänglich sind. Die lange lag-Phase nach Einsetzen der Erstbelichtung benötigt das P730 offenbar dazu, die potentiell aktiven Gene (P730) für das P730 zugänglich zu machen. Die diesbezüglichen Änderungen, vermutlich in der Umgebung der Gene, bleiben auch dann erhalten, wenn P730 verschwindet. P730 scheint also zwei distinkte Funktionen zu haben, die man experimentell trennen kann: Es macht die Gene zugänglich und es aktiviert die betreffenden Gene.

Die Photomodulation der Akkumulationsrate von Ascorbinsure beim Senfkeimling (Sinapis alba L.) durch Phytochrom@@@Photomodulation by phytochrome of the rate of accumulation of ascorbic acid in mustard seedlings (Sinapis alba L.)

SummaryAscorbic acid accumulation in the mustard seedling is controlled by Pfr(active phytochrome). Kinetic studies demonstrate that Pfrexerts a rapid and fully reversible control over the steady state rate of ascorbic acid accumulation. Following the terminology of Weisz (1967) for this type of metabolic control the term “photomodulation by Pfr” is used.—The control by Pfris independent of RNA synthesis. Therefore regulation of gene activity is probably not involved in photomodulation of the rate of ascorbic acid accumulation.—There is only a limited period within which Pfrcan control ascorbic acid accumulation. This period is fixed by the time pattern of “primary” differentiation in the seedling. There is no interaction between photomodulation by Pfrand control by “primary” differentiation of ascorbic acid accumulation.

Eine neue Beweisfhrung fr die Hypothese einer differentiellen Genaktivierung durch Phytochrom 730@@@New evidence in favour of the hypothesis of differential gene activation by phytochrome 730

SummaryPhytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) can be regarded as a prototype of a “positive” photoresponse. “Positive” photoresponses are those which are characterized by an initiation or an increase of biosynthetic or growth processes. The initial lag-phase of this type of photoresponse is relatively long. In the case of anthocyanin synthesis of the mustard seedling it takes about 3 hours after the initial formation of P730, the active phytochrome, until anthocyanin synthesis can be measured (Fig.). This period of time has been called initial (or primary) lag-phase.In earlier papers (e.g. Lange and Mohr, 1965; Mohr, 1966a, b; Schopfer, 1967) we have advanced and supported the hypothesis that the positive photoresponses (in the above sense) of the mustard seedling can be explained by a differential gene activation through P730. Those genes of which the activity can be started or increased by P730 have been called potentially active genes (P730).In order to verify this hypothesis further we have been trying to characterize the processes which occur during the initial lag-phase after the onset of continuous far-red light. There is good evidence (Hartmann, 1966; Clarkson and Hillman, 1967) that under continuous far-red a low but virtually stationary concentration of the active P730 can be maintained in the seedlings tissues over a period of many hours. Consequently we are dealing with steady state conditions of P730 when we investigate the process of anthocyanin accumulation under continuous far-red.In the present paper we present data which indicate that P730 exerts two functions during the initial lag-phase. First, it eliminates a barrier before the potentially active genes insolfar as it makes these genes accessible for the activating action of P730. Secondly, P730 starts the activity of potentially active genes. To maintain gene activity the continuous presence of P730 is required. On the other hand, it a gene has once been “opened” to the action of P730 it remains easily accessible for the activating action of P730 even in the case when P730 disappeared and gene activity ceased for an extended period of time. After the re-appearance of P730 the gene can be activated almost immediately. These conclusions have been derived from the following facts.1.The initial lag-phase after the onset of far-red is always in the order of 3 hours (Fig.). If, however, a seedling which was preirradiated with 12 hours of far-red is kept in darkness for an extended period and is then re-irradiated with far-red no lag-phase for the action of the second irradiation can be found even when anthocyanin synthesis had already ceased during the preceding dark period (Fig.).ZusammenfassungNach Erstbelichtung des Senfkeimlings mit Dauer-Dunkelrot zeigt die Anthocyansynthese, eine typische “positive Photomorphose”, eine mehrere Stunden währende lag-Phase. Bei einer Zweitbelichtung (Programm: Erstbelichtung — längere Dunkelperiode — Zweitbelichtung) fehlt die lag-Phase (Abb.). — Auch die Zweitbelichtung dürfte die Anthocyansynthese über eine Genaktivierung, die eine Enzymsynthese zur Folge hat, auslösen (Tabelle). — Die wichtigsten Konsequenzen: P730, das aktive Phytochrom, kann die Enzymsynthese sehr rasch bewirken, falls die Gene der Aktivierung durch P730 zugänglich sind. Die lange lag-Phase nach Einsetzen der Erstbelichtung benötigt das P730 offenbar dazu, die potentiell aktiven Gene (P730) für das P730 zugänglich zu machen. Die diesbezüglichen Änderungen, vermutlich in der Umgebung der Gene, bleiben auch dann erhalten, wenn P730 verschwindet. P730 scheint also zwei distinkte Funktionen zu haben, die man experimentell trennen kann: Es macht die Gene zugänglich und es aktiviert die betreffenden Gene.

Die Steigerung der durch Phytochrom bewirkten Anthocyansynthese des Senfkeimlings (Sinapis alba L.) durch Chloramphenicol@@@The increase of phytochrome-mediated anthocyanin synthesis in the mustard seedling (Sinapis alba L.) by chloramphenicol

SummaryChloramphenicol (CAP) within a certain range of concentration (about 20–30μg/ml) increases the rate of far-red mediated anthocyanin accumulation in the mustard seedling (Sinapis alba L.) (Fig. 1). The lag-phase after the onset of far-red and the time of termination of anthocyanin synthesis are not influenced by the presence of the antibiotic (Fig. 2). With and without CAP we observe a constant rate of anthocyanin accumulation over a period of at least 24 hours after the lag-phase at all far-red intensities investigated (Fig. 2). The percentage increase of the rate of anthocyanin accumulation which is due to CAP (20 μg/ml) is independent of the far-red intensity applied and always amounts to about 25% (Table). Formally one can conclude that CAP and far-red seem to act as two independent factors in a multiplicative system. On the “molecular” level the observations can possibly be explained as follows: CAP at a concentration of 20 μg/ml inhibits protein synthesis of the plastids. This inhibition leads to an increase of the pool of phenylalanine in the cotyledons. Because the small concentration of CAP does not interfere with protein synthesis (enzyme synthesis) in the cytoplasm, far-red mediated anthocyanin synthesis can proceed normally. Since phenylalanine acts as a precursor of flavonoids the increased pool of this substance will lead to an increase in the rate of anthocyanin accumulation.ZusammenfassungChloramphenicol (CAP) steigert in einem gewissen Konzentrationsbereich (etwa 20–40 μg/ml) die durch Dunkelrot (DR) ausgelöste Anthocyansynthese des Senfkeimlings. Die lag-Phase sowie der Zeitpunkt der Beendigung der Anthocyansynthese werden durch das Antibioticum nicht beeinflußt. Mit und ohne CAP beobachtet man bei allen untersuchten DR-Intensitäten eine konstante Akkumulationsrate an Anthocyan über einen Zeitraum von mindestens 24 Std. —Die Steigerung der Akkumulationsrate durch CAP (20 μg/ml) liegt stets im Bereich von 25%, auch wenn die DR-Intensität auf die Hälfte oder ein Viertel gesenkt wird (Wirkung des CAP=25% · Wirkung des DR). Man kann daraus formal schließen, daß CAP und DR unabhängig voneinander die Anthocyansynthese beeinflussen. “Molekular” läßt sich der Sachverhalt folgendermaßen deuten: CAP hemmt bei einer Konzentration von 20 μg/ml die Proteinsynthese der Plastiden. Dadurch wird der Phenylalanin-pool in den Kotyledonen erhöht. Da diese geringe CAP-Konzentration die cytoplasmatische Proteinsynthese noch nicht hemmt, kann die Anthocyansynthese ungestört ablaufen. Da Phenylalanin als eine Vorstufe der Flavonoidsynthese fungiert, führt das erhöhte Angebot an dieser Substanz zu einer Steigerung der Anthocyansynthese.