Die Wirkung von Actinomycin D auf die phytochromabhängigen Veränderungen des RNS-Gehaltes im Senfkeimling (Sinapis alba L.)

Summary Actinomycin D (10 g/ml) cancels completely the phytochrome-mediated RNA net synthesis in the cotyledons of the mustard seedling whereas RNA net synthesis in the cotyledons of the dark-grown seedling is only partially inhibited (Fig. 1). — In the hypocotyl Actinomycin D of the same concentration lowers the RNA contents in the light (i.e. far-red)-grown seedling as well as in the dark-grown seedling down to the same level (Fig. 2). In the presence of Actinomycin D phytochrome has no significant influence on the RNA contents neither in the cotyledons nor in the hypocotyl (Fig. 1,2).The data support the view that P₇₃₀, the active phytochrome, acts through differential gene activation in the cotyledons and predominantly through differential gene repression in the hypocotyl (cf. Mohr, 1966; Schopfer, 1967a, b). —The data further support the conception that active genes (as defined by Mohr, 1966 and Schopfer, 1967a, b) are much less sensitive towards Actinomycin D than potentially active and repressible genes (cf. Schopfer, 1967a; Mohr and Bienger, 1967).     

Ein Beitrag zur Interpretation der Dunkelrotbande der Hochenergiereaktion bei der Photomorphogenese (Lichtabhängige Anthocyansynthese bei Senfkeimlingen,Sinapis alba L.)

Zusammenfassung Die Anthocyansynthese des Senfkeimlings (Sinapis alba L.) wird über das Phytochrom und eine hypothetische Hochenergiereaktion (=HER) reguliert (Mohr 1957). Das Wirkungsspektrum der HER zeigt Gipfel im Dunkelrot und im Blau.Die vorliegende Arbeit befaßt sich mit der Frage, ob wenigstens die dunkelrote Wirkungsbande der HER auf der Basis des Phytochroms zu deuten sei. Die Resultate (Wirkungsspektrum der HER mit Hellrot-Hintergrund, Intensitäts-Effekt-Kurven, Zeit-Effekt-Kurven) schließen eine befriedigende Deutung auf der Basis der momentan akzeptierten Theorie des Phytochroms aus.Eine längere Vorbestrahlung mit Dunkelrot erhöht die Wirksamkeit einer kurzen Hellrot-Bestrahlung. Eine Vorbestrahlung mit Hellrot hingegen erniedrigt die Empfindlichkeit des Systems für Dunkelrot. Auch die Deutung dieser Daten erfordert eine Modifikation der Theorie des Phytochroms. Im Anschluß an eine diesbezügliche Hypothese von Hartmann (1965) für die HER des Lactuca sativa-Keimlings wird die Annahme gemacht, daß zumindest die dunkelrote Wirkungsbande der HER auf der Basis des Phytochromsystems gedeutet werden kann, falls die in vivo-Destruktion von P₇₃₀ in die Theorie einbezogen wird. Für eine befriedigende quantitative Theorie der HER des Senfkeimlings auf der Basis einer modifizierten Phytochromtheorie sind aber weitere Daten und Überlegungen erforderlich.

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A contribution to interpret the far-red action band of the high energy reaction of photomorphogenesis (light dependent synthesis of anthocyanin in mustard seedlings, Sinapis alba L.)

Summary Anthocyanin synthesis in mustard seedlings is known to be controlled by phytochrome and by a hypothetical high energy reaction (Mohr 1957). This high energy reaction (=HER) is characterized by an action spectrum with peaks in the far-red and in the blue range of the visible spectrum (Fig. 2). Photosynthesis is not involved (Bertsch and Mohr 1965).The present paper is concerned with the problem of whether at least the far-red action band of the HER could be explained on the basis of phytochrome. It is evident, however, that the action spectrum of the HER elaborated with simultaneous red background irradiation (Fig. 2), the irradiance-response curves (Fig.3) and the timeresponse curves (Fig. 4) are hardly to be explained on the basis of the presently accepted phytochrome theory (e. g. Hendricks 1964).Red light is only slightly effective in anthocyanin synthesis of mustard seedlings when brief exposures to red are used in order to once saturate the photostationary state of phytochrome (Fig.5). If, however, brief exposures to red are preceded by long exposures to far-red, the red is much more effective (Fig.6). Long exposures to far-red increase the sensitivity of the system to red (Table 1). — On the other hand, a pretreatment with long exposures to red will decrease considerably the sensitivity of the system to far-red (Fig. 7, Table 1). These data, too, cannot be understood solely on the basis of the conventional phytochrome theory.It is assumed, following a recent hypothesis by Hartmann (1965) for hypocotyl lengthening in lettuce seedlings (Table 2), that at least the far-red band of action of the HER can eventually be understood on the basis of phytochrome if the in-vivo destruction of phytochrome 730 (Butler 1964) is taken into account. This assumption is supported by recent experiments in which anthocyanin synthesis in the mustard seedling was studied under steady state conditions of irradiation (Mohr, Wagner and Hartmann 1965), but more data and more reasoning will be required before the explanation of the HER on the basis of phytochrome will be satisfactorily established.

     

Weitere Untersuchungen zur phytochrominduzierten Akkumulation von Ascorbinsäure beim Senfkeimling (Sinapis alba L.)

Summary The accumulation of ascorbic acid (AS) in the young mustard seedling is greatly increased by the action of the active form of phytochrome (P₇₃₀) (see Schopfer, 1966). There is no photosynthesis in continuous far-red light, which was used throughout the experiments. The phytochrome-mediated increase in AS approximately parallels the synthesis of anthocyanin in the seedling, although the onset of AS-accumulation precedes the anthocyanin synthesis by 2–3 hours (Fig. 4 and 5).—The action of P₇₃₀ increases the amount of AS in every part of the seedling (cotyledons, hypocotyl, radicula) (Fig. 1–3). This increase in AS parallels the formation of P₇₃₀ rather than the different growth responses of these organs (enlargement of the cotyledons, inhibition of hypocotyl and radicula lengthening). The lag in AS-accumulation after onset of far-red irradiation is the same in all 3 parts of the seedling (about 1 hour under the experimental conditions; Fig. 4).—Actinomycin D (10 g/ml), which strongly inhibits anthocyanin synthesis (Fig. 9 and 10), has no corresponding effect on P₇₃₀-dependent increase in AS-accumulation (Fig. 7 and 8). This result support the hypothesis that already active genes are only slightly influenced by actinomycin D (see Lange and Mohr, 1965). It also shows that, in contrast to its role in anthocyanin synthesis, P₇₃₀ probably does not act by initiating potentially active genes in the case of AS-accumulation. — A dark synthesis of anthocyanin in the cotyledons can be obtained by application of AS to the seedlings (Fig. 11). Glucose and sucrose are ineffective in this respect (Table 2). The effect of AS-feeding on anthocyanin synthesis can be inhibited by actinomycin D in very much the same way as light induced anthocyanin synthesis is inhibited (Table 3). — Also the RNA content of the cotyledons is increased by feeding AS in the dark (Table 4).These results are in line with the earlier suggested hypothesis (see Schopfer, 1966) that increase in AS-accumulation is a very early event in the mediation of some positive photoresponses, e.g. anthocyanin synthesis. According to the hypothesis of Mohr (1966 a, b) it has been concluded that AS functions as a part of the signal chain of phytochrome-mediated photomorphogenesis. This signal chain is thought to start differential gene activation to bring about positive photoresponses.     

Die Regulation der PAL-Aktivität durch Phytochrom in Senfkeimlingen (Sinapis alba L.)

Summary The present paper is a contribution to the molecular analysis of photomorphogenesis. L-phenylalanine ammonia-lyase (=PAL) (EC 4.3.1.5) has been used as a model system to demonstrate that enzyme synthesis, enzyme inactivation and gene repression are important in determining the response of a particular enzyme to phytochrome.The level of PAL in the mustard seedling is controlled by P_fr (the active form of phytochrome) in a characteristic manner which is illustrated in Fig. 1. The seedlings were irradiated with continuous standard far-red light. Long time irradiation with far-red will maintain a low but virtually constant level of the effector molecule P_fr in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of P_fr will instantly decrease and will eventually cease probably within the order of an hour (cf. Karow and Mohr, 1969). The approach followed in the present paper has been to turn off the far-red light after varying periods and follow the enzyme kinetics in darkness (Fig. 2). The main results can be summarized as follows: The far-red kinetics of PAL (Fig. 1) can be explained as the result of three processes, namely, Pfr-mediated enzyme synthesis, inactivation of PAL by an inactivator, and eventual repression of enzyme synthesis.—During the period 1.5–12 hrs after the onset of far-red only enzyme synthesis occurs. Then enzyme inactivation comes into play while enzyme synthesis continues at a constant rate (Fig. 3). This antagonism of synthesis and inactivation leads to a true steady state which is observed between about 24 and 27 hrs after the onset of far-red. After this period the rate of enzyme synthesis decreases and as a consequence, inactivation dominates. 36 hours after the onset of far-red the P_fr-mediated PAL synthesis is hardly dtectable. The results of secondary irradiations with far-red (Fig.4) indicate that the inactivator of PAL does not have any direct influence on PAL synthesis. The kinetics in darkness (Fig.1,2) can best be understood by assuming that a certain enzyme level represented by the plateau cannot be overcome in the dark. The overshoot response which is obvious in the enzyme kinetics immediately after the cessation of far-red (Fig. 2) cannot be explained readily in molecular terms.

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PAL=Phenylalaninammoniumlyase (EC 4.3.1.5).

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Diese Arbeit ist Herrn Professor H. Borris, Greifswald, mit guten Wünschen zum 60. Geburtstag gewidmet.     

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

The control by phytochrome of hypocotyl elongation of light-grown Cucumis sativus L. after a white-light period was examined. The farred-absorbing form of phytochrome inhibits hypocotyl elongation. The response to phytochrome photostationary state () is not linear; all values of from 0.004 to 0.13 promote growth maximally, in the range of values of from 0.13 to 0.22 there is a linear growth response, between values of of 0.22 and 0.35 there is again no differential effect, and for values above 0.35 there is a strong (near linear) effect of on elongation. A kinetic examination of events following the white-light period shows that the major recovery from the photoperiod requires 8.5 h of darkness. End-of-day far-red treatment produces a very different response pattern, with a minor growth stimulation within 28 min of treatment followed by a major effect after 80 to 90 min. Three hours after far-red treatment there is a transient decline in growth rate which persists for about 2 h. Over the whole time course there is a great stimulation of growth rate compared with the controls. A similar growth-rate pattern also occurs if the end-of-day is 0.48, although the magnitude of the growth stimulation is less. Two components are affected by end-of-day , namely the time at which growth recovers and the subsequent growth rate. In the long term, the latter accounts for most of the differences in elongation growth. The dark recovery when only the hypocotyl is irradiated requires 4 h, but end-of-day far-red treatment reduces this to about 1.5 h. The persistence of the far-red-absorbing form of phytochrome for many hours in darkness in these light-grown plants is also demonstrated.Abbreviations and symbols D darkness - FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light - WL white light (from fluorescent lamps) - photostationary state of phytochrome - _c calculated      

Der Lichteinfluss auf die Haarbildung am Hypokotyl vonSinapis alba L.

Summary In an earlier paper (Mohr 1957) it was described that the formation of anthocyanin and the inhibition of lengthening of the hypocotyl of dark grown seedlings (Sinapis alba) is governed by the action of two photomorphogenic action systems. The one system is the well known reversible red far-red reaction system (low energy reaction), the other one is a high energy reaction system which can be called — in reference to the action peaks in the far-red and in the blue — blue far-red reaction system. The chemical nature of the absorbing pigments is still unknown.In the present paper another photomorphogenic response of the young dark grown seedlings ofSinapis alba, the light dependent formation of unicellular hairs from epidermal cells of the hypocotyl, has been investigated. It has been shown that this response is also governed by these two reaction systems. These systems have been physiologically separated. Experiments have been presented which evaluate the importance of the assumption that the red absorbing pigment of the reversible red far-red pigment system is reformed from a precursor when the pigment present before irradiation is transformed into the far-red absorbing pigment by an irradiation with red.

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Mit 3 Textabbildungen     

Der Einfluss von Phytochrom auf die Stationären Konzentrationen von Ascorbinsäure und Dehydroascorbinsäure beim Senfkeimling (Sinapis alba L.)

Zusammenfassung Der Senfkeimling (Sinapis alba L.) synthetisiert auch im Dunkeln beträchtliche Mengen an Ascorbinsäure. Durch Licht kann der Ascorbinsäure-Gehalt der Keimlinge stark erhöht werden. Dieser Lichteinfluß ist auf die Funktion von Phytochrom zurückzuführen. Die Photosynthese hat keinen wesentlichen Anteil an der Lichtwirkung. Die Konzentration an Dehydroascorbinsäure ist im Verhältnis zur Ascorbinsäurekonzentration stets sehr gering (5–8% der Konzentration an Totalascorbat) und wird vom Phytochrom nicht beeinflußt.Wenn die Funktion von Phytochrom bei den positiven Photomorphosen unter dem Aspekt der differentiellen Genaktivierung (vgl. Mohr, 1966) betrachtet wird, kann die folgende Arbeitshypothese aufgestellt werden: Die durch Phytochrom 730 induzierte Ascorbinsäureakkumulation führt im Bereich von potentiell aktiven Genen zu einer Spaltung gewisser DNS-Histon-Komplexe. Dadurch wird die Synthese von m-RNS an diesen Genen ermöglicht, was schließlich zur Ausbildung der positiven Photomorphosen führt. Die Argumente, welche zur Zeit für eine Funktion der Ascorbinsäure bei der Regulation der Genaktivität sprechen, werden kurz diskutiert.

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The control by phytochrome of the contents of ascorbic acid and dehydroascorbic acid in the mustard seedling (Sinapis alba L.)

Summary A dark grown seedling of white seeded mustard (Sinapis alba L.) contains an appreciable amount of ascorbic acid. The content of ascorbic acid, however, will strongly increase under the influence of light. This effect is due to phytochrome. Photosynthesis is not involved under our experimental conditions.The content of dehydroascorbic acid is always very low compared to ascorbic acid (5–8% of total ascorbate). Phytochrome does not influence this relation.The lag-phase of the phytochrome induced increase in ascorbic acid accumulation is remarkably short, about 1 hour after the onset of light compared to about 4 hours for phytochrome induced anthocyanin synthesis under our conditions.This is the shortest lag-phase we have observed hitherto in the case of positive photoresponses (Mohr, 1966).If we assume that the function of phytochrome in the case of positive photoresponses involves a differential gene activation of potentially active genes (Mohr, 1966) the following working hypothesis can be advanced: phytochrome induced accumulation of ascorbic acid will lead to a separation of DNA-histone complexes in the range of potentially active genes. This makes possible the DNA-dependent synthesis of m-RNAs at those sites which are lastly responsible for the initiation of positive photoresponses. — Arguments are briefly considered which support the view that ascorbic acid exerts a function in connection with the regulation of gene activity.

     

Die Steuerung der Antheridienbildung in Polypodium crassifolium L. (Pessopteris crassifolia Underw. and Maxon) durch Licht

Summary In Polypodium crassifolium, light controls the induction of antheridium formation in contrast to hormonal induction in other fern species.Antheridium formation is caused by an exposure to darkness or far-red. The minimum duration of this treatment required to bring about antheridium formation depends on the length of preillumination with white light.Red light interruptions of 5 min (6,6 E cm^-2 sec^-1) at 24 h intervals applied during the whole dark period extinguish antheridium induction. Red light inhibition is cancelled by a succeeding irradiation with far-red.Inhibitors of protein and nucleic acid synthesis do not block antheridium induction. Actinomycin-D inhibits the formation of spermatogenic tissue.The results presented indicate a control of antheridium formation in Polypodium crassifolium via a negative photoresponse (Mohr, 1966). So ist appears highly improbable that this differentiation process is based on a specific gene activation in the sense of Jacob and Monod.     

The relationship between phytochrome-photoequilibrium and Development in light grown Chenopodium album L.

Chenopodium album seedlings were grown in light environments in which supplementary far-red light was mixed with white fluorescent light during various parts of the photoperiod. Both the logarithmic rate constant of stem extension and the leaf dry weight: stem dry weight ratio were linearly related to estimated phytochrome photoequilibrium () in each treatment regime. These data are taken to be indicative of a functional link between phytochrome and development in the green plant. A layer of chlorophyllous tissue only affected the linearity between calculated and the logarithmic stem extension rate at high chlorophyll concentrations, whilst even low concentrations-equivalent to the levels found in stem tissue-caused a significant shift in measured . End-of-day supplementary far-red (FR) light induced between 0–35 per cent of the response elicited by all-day supplementary FR, whilst daytime supplementary FR (with a white fluorescent light end-of-day treatment) induced approximately 90 per cent. The ecological significance of this difference is discussed with respect to shade detection.Paper 7 in the series The function of phytochrome in the natural environment [for paper 6 see McLaren, J.S., Smith, H., Plant, Cell and Environment 1, 61–67, 1978]     

Zur Regulation der RNS-Synthese durch Phytochrom bei der Photomorphogenese des Senfkeimlings (Sinapis alba L.)

Summary P₇₃₀, the active phytochrome, increases the rate of RNA synthesis (Table) and the RNA contents in the cotyledons of the mustard seedling (Sinapis alba L.) (Fig. 1) whereas the RNA contents in the hypocotyl is decreased under the influence of P₇₃₀ (Fig. 2).—It takes about 6 hours until changes in the RNA contents-which must be attributed to the formation of P₇₃₀—can be measured after the onset of light (continuous far-red). Since the lag-phases of positive photoresponses in the cotyledons and negative photoresponses in the hypocotyl (Mohr, 1966) are in general much shorter than 6 hours, the changes of the RNA contents of the organs cannot be regarded as being directly connected with the formation of characteristic positive photoresponses such as anthocyanin synthesis, induced enzyme synthesis, ascorbic acid synthesis, etc., or negative photoresponses such as inhibition of hypocotyl lengthening.We have rather to conclude that the changes of RNA contents are secondary adaptations of the organs which lead to an increase (cotyledons) or decrease (hypocotyl) of protein synthesizing capacity of the cells and tissues. The P₇₃₀-dependent increase of bulk RNA in the cotyledons is probably due to a differential gene activation and the P₇₃₀-dependent decrease of bulk RNA in the hypocotyl is due to a differential gene repression. The causalities of these processes are possibly complex.The hypothesis of differential gene activation or repression by P₇₃₀ (Mohr, 1966; Schopfer, 1967a, b) is not disproved by these results. We have rather to reach a conclusion which has already been suggested by other data (e.g. Karow and Mohr, 1966), namely, that positive as well as negative photoresponses are due to changes in the activity of a limited (possibly small) number of enzymes. Correspondingly changes in only a minute amount of the total RNA are directly involved in the formation of photoresponses. These changes cannot be detected by following RNA contents.—It seems to be of great interest, however, that P₇₃₀ eventually brings about strong tissue specific changes in the RNA contents per cell as described in the present paper.     

Die histologischen Vorgänge während der lichtabhängigen Schliessung und Öffnung des Plumulahakens bei den Keimlingen vonLactuca sativa L.

Summary In a preceding paper the following facts have been shown (Mohr undNoblé 1960): Dark grown seedlings ofLactuca sativa L. var.Grand Rapids (tip burn resistant strain) do not form an appreciable plumular hook. But a normal plumular hook, as is formed for instance byPhaseolus vulgaris in complete darkness, can be induced by red light. This formation of the plumular hook is controlled by the phytochrome system. — The hook which has been closed by red radiation can be reopened by light. In this respect only the high energy reaction of photomorphogenesis is effective. This reaction system shows peaks of action in the blue and in the far-red part of the visible spectrum (cf.Mohr 1962).The present paper deals with the investigation of the anatomical and cellular changes which lead to hook formation and reopening. The movements of the hook always take place in the cotyledonary plane. Bending of the hook accurs because the cells on the potentially outer side of the future hook increase their rate of elongation, unbending of the hook is due to a rapid increase in rate of elongation of the cells on the inner side of the closed hook. Cell dividions are apparently not involved either in the bending or in the unbending of the hook. It is concluded, that the apical part of the hypocotyl is a highly complex organ the potentialities of which can be described only with the help of physiological experimentation. The conventional anatomical or cytological approaches are not adequate to describe in detail the pattern of differentiation which must be present in the apical part of the hypocotyl according to the reactions which can be induced by light.

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Mit 13 Textabbildungen

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Als Plumulahaken (=plumular hook) bezeichnen wir die auffällige Einkrümmungszone im Apikalbereich einer Keimpflanze. Ein knapper und klarer Begriff hat sich offenbar in der deutschen Literatur nicht durchgesetzt. AuchTroll (1959) spricht nur allgemein von einer apikalen Krümmung des Keimsprosses. Bei dem Plumulahaken derLactuca-Keimlinge handelt es sich um eine Einkrümmung des apikalen Hypokotylbereichs.     

Absence of fluence rate dependency of phytochrome modulation of stem extension in light-grown Sinapis alba L.

In background white light, supplementary far-red (_max 700 nm) is an order of magnitude less effective than supplementary far-red (_max 739 nm) in the stimulation of stem extension in Sinapis alba. The relationship between phytochrome photoequilibrium and extension rate increase for the two supplementary far-red treatments is, however, very similar. This evidence indicates that phytochrome cycling is not involved in the phytochrome control of stem extension in light-grown Sinapis alba and that the response to supplementary far-red light is not fluence rate (irradiance) dependent.Abbreviations Pfr far-red absorbing form of phytochrome - the phytochrome photoequilibrium (Pfr/Ptotal)     

Vom Einfluß des Phytochroms auf die Xylemdifferenzierung im Hypokotyl des Senfkeimlings (Sinapis alba L.)

Zusammenfassung P₇₃₀, das aktive Phytochrom, bewirkt eine vermehrte Bildung von Gefäßen (Tracheen und Tracheiden) im Hypokotyl des Senfkeimlings. Das Differenzierungsmuster der Leitbündel und der Verlauf der Leitbahnen sind im belichteten und im etiolierten Keimling gleich. Es wird geschlossen, daß auch bezüglich der Ausbildung der Leitbahnen das P₇₃₀ lediglich im Rahmen einer sekundären Differenzierung als Auslöser wirkt. Die primäre Differenzierung (vgl. Wagner und Mohr, 1966 b) wird durch P₇₃₀ offenbar nicht beeinflußt.

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Phytochrome-mediated control of xylem differentiation in the hypocotyl of the mustard seedling (Sinapis alba L.)

Summary P₇₃₀, the active phytochrome, causes an increased formation of xylem elements (tracheids and vessel elements) in the hypocotyl of the mustard seedling (Figs. 3,4). On the other hand, the pattern of differentiation of the bundles and the course of the bundles within the hypocotyl (Figs. 1,2) are the same in etiolated as well as in illuminated seedlings.—It has been concluded that in connection with bundle differentiation P₇₃₀ acts only as a trigger at the level of secondary differentiation. The pattern of differentiation is laid down in the course of primary differentiation which apparently is not influenced by P₇₃₀. The same problem has been dealt with more in detail in a foregoing paper (Wagner and Mohr, 1966b).

     

Die Hemmung der Phytochrominduzierten Photomorphogenese („Positive” Photomorphosen) des Senfkeimlings (Sinapis alba L.) Durch Actinomycin D und Puromycin

Zusammenfassung Die positiven Photomorphosen Öffnung des Hypokotylhakens und Entfaltung der Kotyledonen können ganz ähnlich wie die phytochrominduzierte Anthocyansynthese und andere positive Photomorphosen durch Actinomycin D und Puromycin gehemmt werden. Man kann daraus schließen, daß diese beiden photomorphogenetischen Reaktionen des Senfkeimlings ebenfalls durch eine von P₇₃₀ über eine Signalkette ausgelöste Aktivierung von potentiell aktiven Genen veranlaßt werden.

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The inhibition of phytochrome-mediated photomorphogenesis (positive photoresponses) by actinomycin D and puromycin in the mustard seedling (Sinapis alba L.)

Summary The many photochrome-mediated photoresponses of a seedling (Sinapis alba L., white seeded mustard) can be divided into 3 categories: positive, negative, and complex photoresponses. Positive photoresponses are those which are characterized by an initiation or a promotion of biosynthetic or growth processes (Mohr, 1966b). Phytochrome-mediated anthocyanin synthesis is the prototype of a positive photoresponse. It has been shown in previous papers (e.g. Lange and Mohr, 1965; Mohr et al., 1965) that positive photoresponses can be specifically inhibited by actinomycin D and puromycin. It has been concluded that in the case of positive photoresponses P₇₃₀ (the active phytochrome) exerts its function through differential gene activation.—In the present paper it has been demonstrated that phytochrome-mediated positive photoresponses of the mustard seedling like opening of the hypocotylar hook and unfolding of the cotyledons can be inhibited by relatively low doses of actinomycin D and puromycin in very much the same way as anthocyanin synthesis or cotyledon enlargement is inhibited. It has been concluded that in these cases too the action of P₇₃₀ must be attributed to an activation of potentially active genes in the manner postulated on the basis of the data on anthocyanin synthesis.

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Die Arbeit wurde durch Sachbeihilfen der Deutschen Forschungsgemeinschaft und der Stiftung Volkswagenwerk (an Prof. Mohr) ermöglicht.     

Sequential control of phytochrome-mediated synthesis de novo of β-amylase in the cotyledons of mustard (Sinapis alba L.) seedlings

In the cotyledons of mustard (Sinapis alba L.) seedlings irradiated from the time of sowing with continuous red light, the photoreversibility of the phytochrome-mediated increase in -amylase activity (EC 3.2.1.2) is lost 36 h after sowing (coupling point). However, the induced increase of -amylase activity cannot be detected before 46 h after sowing (starting point). Density labeling with deuterium oxide shows that the increase of enzyme activity in light and darkness coincides precisely with the synthesis of -amylase protein. Thus, phytochrome mediates an increase of -amylase synthesis de novo. Since there is no turnover detectable by density labeling, it is concluded that -amylase of mustard cotyledons is a physiologically stable enzyme (half-life >5 d). The 10-h time gap between loss of photoreversibility and onset of light-induced -amylase synthesis points to a relatively stable regulatory element within the signal chain (transmitter) which links -amylase synthesis to the primary action of phytochrome. A 12-h lag between the cessation of phytochrome action and the cessation of induced -amylase synthesis indicates a limited lifetime of the transmitter (about 12 h). The effect of this result on the interpretation of the coupling point is discussed.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome     

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling growth of pine (Pinus sylestris L.) seedlings

Photomorphogenesis is a conspicuous feature in conifers. In the case of the shade-intolerant Scots pine (Pinus sylvestris L.), control of stem growth by light is well expressed at the seedling stage and can readily be studied. The present data show that hypocotyl growth is controlled by the far-red-absorbing form of phytochrome (Pfr). However, the Scots pine seedling requires blue or ultraviolet (UV-A) light to become fully responsive to Pfr. Blue/UV-A light has no direct effect on hypocotyl growth and its action appears to be limited to establishing the responsiveness of the seedling to Pfr. This type of coaction between phytochrome and blue/UV-A light has been observed previously in a number of angiosperm seedlings. With regard to the high irradiance reaction of phytochrome in long-term far-red light the pine seedling deviates totally from what has been observed in etiolated angiosperms since continuous far-red light has no effect on stem growth.Abbreviations B light of wavelength between 500 and 400 nm - FR standard far-red light - HIR high irradiance reaction of phytochrome - R high-fluence-rate red light (_R = 0.8) - RG9-light long-wavelength far-red light defined by the properties of the Schott RG9 glass filter (_RG₉<0.01) - = Pfr/Ptot wavelength-dependent photoequilibrium of the phytochrome system (far-red-absorbing form of phytochrome/total phytochrome) - UV-A near ultraviolet light of wavelength between 400 and 320 nm - W white light Research supported by a grant from the Deutsche Forschungsgemeinschaft (Schwerpunkt Physiologie der Bäume).     

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

An interaction is demonstrated between the effects of phytochrome and cryptochrome (the specific blue-light photoreceptor) in the inhibition of hypocotyl elongation of light-grown cucumber (Cucumis sativus L.) cv. Ridge Greenline seedlings. At certain fluence rates of blue light the total inhibition response is greater than the sum of the separate responses to each photoreceptor. The threshold for response to blue light is reduced at least 30-fold by additional red-light irradiation. The synergistic effect is demonstrated for two different fluence rates of red light. Synergism is mediated by phytochrome in both the cotyledons and the hypocotyl.Abbreviations and symbols BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light - photostationary state of phytochrome - _c calculated      

Die hemmung der Phytochrom-induzierten Anthocyansynthese durch puromycin und 2-thiouracil

Zusammenfassung Die durch Phytochrom 730 bewirkte Anthocyansynthese des Senfkeimlings kann durch Puromycin und 2-Thiouracil gehemmt werden. Im Gegensatz zu Actinomycin D (Lange u Mohr, 1965) hemmen diese Substanzen die Anthocyansynthese auch dann fast völlig, wenn sie erst längere Zeit, z. B. 12 Std. nach Lichtbeginn appliziert werden. Der Effekt von 2-Thiouracil kann aufgehoben werden, wenn man die Keimlinge in ein Medium mit Uracil oder Wasser bringt. Auch die gleichzeitige Applikation von Uracil und 2-Thiouracil führt zu einer zumindest partiellen Aufhebung des Hemmeffektes von 2-Thiouracil. Aus den Kinetiken kann man folgern, daß die Lebensdauer des kurzlebigsten Enzyms, das an der Anthocyansynthese beteiligt ist, in der Größenordnung von 6 Std liegt. Der Schluß erscheint aufgrund der Abb. 1. u. 2 berechtigt, daß die Synthese dieses Enzyms über P₇₃₀ reguliert wird. — Die Wirkung von 2-Thiouracil kann in unserem Fall offensichtlich nicht nur damit gedeutet werden daß 2-Thiouracil in RNS engebaut wird und dadurch falsche RNS entsteht. Man muß vielmehr annehmen, daß das 2-Thiouracil auch eine direkte, kompetitive Enzymhemmung verursacht.

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The inhibitory effect of puromycin and 2-thiouracil on the phytochrome-mediated synthesis of anthocyanin

Summary The phytochrome-mediated anthocyanin synthesis of the mustard seelding, Sinapis alba L., can be stopped by the application of relatively low concentrations of puromycin and 2-thiouracil. While actinomycin D, which has been investigated in a previous paper (Lange and Mohr, 1965), will block phytochrome-induced anthocyanin synthesis only if it is applied before or at the onset of light, puromycin and 2-thiouracil will stop anthocyanin synthesis even if they are applied at a later stage, e.g., 12 hours after the onset of light. — The effect of 2-thiouracil can be reversed if the seedlings are transferred to a medium containing uracil or to blank water. The simultaneous application of 2-thiouracil and uracil also leads to at least a partial reversal of the 2-thiouracil effect. — The kinetics of the puromycin inhibition (Fig. 2) indicate that the enzyme with the shortest life-time among those enzymes which are involved in anthocyanin production has a life-time in the order of 6 hours. One may reasonably conclude on the basis of Fig. 1 and 2 that synthesis of this particular enzyme is controlled by phytochrome 730. — The effect of 2-thiouracil (Fig. 3) cannot — in our case at least — be understood by only assuming that 2-thiouracil will be incorporated into RNA and thus will lead to the formation of wrong RNA. Inhibition by 2-thiouracil is much faster than inhibition by puromycin (Fig. 2,3). We have rather to conclude that 2-thiouracil may exert its effect mainly through a direct competitive enzyme inhibition.

     

Die Wirkung von Phytochrom und Actinomycin D auf die Chlorophyll a-Synthese von Senfkeimlingen (Sinapis alba L.)

Zusammenfassung Eine Vorbestrahlung der etiolierten Senfkeimlinge mit 4 Std Dunkelrot bewirkt eine Steigerung der Chlorophyll a-Synthese im Weißlicht. — Andererseits wird die Chlorophyll a-Synthese bereits durch relativ niedrige Actinomycin D-Konzentrationen gehemmt oder verzögert. — Die hemmende Wirkung von Actinomycin D ist — ausgedrückt in Prozent Hemmung — mit und ohne Vorbelichtung genau dieselbe. Auch die übrigen Daten deuten darauf hin, daß Actinomycin D nicht auf die Protochlorophyllsynthese als solche wirkt, sondern vielmehr die Synthese bestimmter Strukturproteine in den Plastiden beeinträchtigt. —Die Daten der Arbeit werden genphysiologisch gedeutet. Der wesentliche Punkt dabei ist, daß aktive Gene (z.B. jene, welche die Enzyme der bekanntlich auch im Dunkeln ablaufenden Protochlorophyllsynthese codieren) relativ unempfindlich gegenüber Actinomycin D sind; potentiell aktive Gene hingegen (z.B. jene, die einige nur im Licht entstehende spezifische Strukturproteine der Plastiden codieren) scheinen sehr viel empfindlicher gegenüber Actinomycin D zu sein. Diese Schlüsse stehen im Einklang mit einer früher geäußerten Hypothese (Lange und Mohr, 1965) und mit den Daten von Schopfer (1966) über die Regulation der Ascorbatsynthese im Senfkeimling durch Phytochrom und Actinomycin D.

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The action of phytochrome and actinomycin D on chlorophyll a formation in mustard seedlings (Sinapis alba L.)

Summary In the mustard seedling chlorophyll a synthesis under white light is enhanced by a pretreatment with far-red which maintains a low but virtually stationary concentration of active phytochrome (=P₇₃₀) (Fig. 1) during the period of irradiation (4 hours). — On the other hand chlorophyll a synthesis is inhibited or delayed by relatively low concentrations of Actinomycin D(=Act) (Fig. 2)The inhibitory action of Act (on a percent basis) is exactly the same with and without a far-red pre-irradiation (Fig. 3). Act in relatively low doses (5 or 10 g/ml) greatly extends the lag-phase of chlorophyll synthesis; however, these doses do not influence the effect of the far-red pretreatment on the rate of chlorophyll synthesis when it finally takes place (Fig.4,5,6). The data presented in this paper indicate that Act does not inhibit protochlorophyll synthesis as such; we have rather to conclude that Act inhibits the de novo synthesis of some specific structural proteins which are prerequisites of chlorophyll accumulation and maintenance in the plastids (Table 1). Synthesis of these structural proteins seems to be under the control of phytochrome too.It is concluded that those genes which are already in function are relatively resistant to Act (e. g. those genes which are needed for protochlorophyll synthesis) whereas potentially active genes (e. g. those which code some specific structural proteins of the plastids) are very sensitive to Act. —A similar conclusion has been reached in an earlier paper in connection with phytochrome-induced antocyanin synthesis (Lange and Mohr, 1965). Our argumentation is further supported by Schopfer's data on control of ascorbate synthesis in the mustard seedling by phytochrome and Act (Schopfer, 1966).