Manfred Weidner

Seasonal variation of enzyme activities in Laminaria hyperborea

The patterns of seasonal variation of enzyme levels in the brown alga Laminaria hyperborea (Gunn.) Fosl. have been investigated for the following enzymes: Ribulosebisphosphate-carboxylase (EC 4.1.1.39), phosphoenolpyruvate-carboxykinase (EC 4.1.1.32), glyceraldehyde-3-phosphate-dehydrogenase (NADP dep., EC 1.2.1.12), malate-dehydrogenase (NAD dep., EC 1.1.1.37), L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1), and mannitol-l-phosphate-dehydrogenase (EC 1.1.1.17). The first four enzymes exhibit a circannual periodicity, characterized by a pronounced ‘spring-maximum’ of enzyme activity in April and May. As a consequence, the phylloid can maintain high metabolic rates from early spring on, although water temperature has then only slightly risen above the annual minimum. This findings is discussed in relationship to the growth- and developmental cycle of L. hyperborea and to the seasonal variation of photosynthesis and light-independent CO2-fixation. The seasonal pattern, outlined above, correlates well with the circannual fluctuations of the nitrogen content of the sea and with the variation of the internal nitrogen- and nitrate-content of the alga. This coincidence may indicate that nitrogen levels play an important role in the regulation of enzyme activities and, hence, the metabolic capacities of L. hyperborea.

Der DNS-Gehalt von Kotyledonen und Hypokotyl des Senfkeimlings (Sinapis alba L.) bei der phytochromgesteuerten Photomorphogenese

DNA contents and accordingly cell numbers of cotyledons and hypocotyl of the mustard seedling were virtually constant during the experimental period (between 36-60 hours after sowing) in the dark as well as under the influence of P730, the active phytochrome (Table).-Therefore the phytochromemediated increase of the RNA and protein contents (WEIDNER et al., 1965) must be understood as an increase of RNA and protein per cell. This fact is in agreement with the conception of differential gene activation mediated by P730 (MOHR, 1966). The virtually constant DNA contents during the period of time which is regularly used for experimentation on photomorphogenesis in our laboratory (36-60 hours after sowing; MOHR, 1966) is an important prerequisite for comparing biochemical data under the point of view of differential gene activation, e.g. in kinetical studies in the dark and under continuous far-red light.SummaryDNA contents and accordingly cell numbers of cotyledons and hypocotyl of the mustard seedling were virtually constant during the experimental period (between 36–60 hours after sowing) in the dark as well as under the influence of P730, the active phytochrome (Table).—Therefore the phytochromemediated increase of the RNA and protein contents (Weidner et al., 1965) must be understood as an increase of RNA and protein per cell. This fact is in agreement with the conception of differential gene activation mediated by P730 (Mohr, 1966). The virtually constant DNA contents during the period of time which is regularly used for experimentation on photomorphogenesis in our laboratory (36–60 hours after sowing; Mohr, 1966) is an important prerequisite for comparing biochemical data under the point of view of differential gene activation, e.g. in kinetical studies in the dark and under continuous far-red light.ZusammenfassungDer DNS-Gehalt und somit auch die Zellzahl von Kotyledonen und Hypokotyl des Senfkeimlings sind zwischen 36 und 60 Std nach Aussaat sowohl im Dunkel als auch im Dunkelrot weitgehend konstant. Die durch Phytochrom bewirkte Zunahme des RNS-und Proteingehaltes (Weidner u. Mitarb., 1965) muß deshalb als eine RNS-bzw. ProteinZunahme pro Zelle aufgefaßt werden. Dieser Befund stützt die Vorstellung einer differentiellen Genaktivierung durch P730 (z.B. Mohr, 1966).—Die weitgehend konstante Zellzahl von Hypokotyl und Kotyledonen während des von uns verwendeten Experimentierzeitraumes ist eine wichtige Voraussetzung für die Vergleichbarkeit biochemischer Daten, z.B. bei kinetischen Studien (vgl. Mohr, 1966).

Lag-phases in phytochrome-mediated enzyme synthesis (PAL)

SummaryIn experiments with the mustard seedling (Sinapis alba L.) it was confirmed that in the case of “secondary irradiation” induction of PAL by phytochrome is a very rapid process. The lag-phase after the onset of light is too brief to be detected. However, the data of other investigators, who found extended “secondary” lag-phases in their experimental material, can be imitated with the mustard seedling. We explain why these investigators were not able to eliminate the “secondary” lag-phase.

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

SummaryThe 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 Pfr (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 Pfr in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of Pfr 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 Pfr-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.

Phosphoenolpyruvat-Carboxykinase und Ribulose-1,5-Diphosphat-Carboxylase von Laminaria hyperborea (Gunn.) Fosl.: Das Verteilungsmuster der Enzymaktivitäten im Thallus

High activities of phosphoenolpyruvate-carboxykinase (EC 4.1.1.32) have been found in fronds of the brown alga Laminaria hyperborea (Gunn.) Fosl. The distribution pattern of this enzyme in the newly formed and in the old phylloid (last years production) and in the cauloid was investigated and compared with that of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39). Maximum activity of both enzymes is located in the growing region of the new phylloid. In the other parts of the frond, enzyme activity is only about one tenth of the values estimated for the intercalary meristem. The activity of PEP-carboxykinase: RuDP-carboxylase shifts from approxymately 9 in the growing region to only 3 in the old phylloid.SummaryHigh activities of phosphoenolpyruvate-carboxykinase (EC 4.1.1.32) have been found in fronds of the brown alga Laminaria hyperborea (Gunn.) Fosl. The distribution pattern of this enzyme in the newly formed and in the old phylloid (last years production) and in the cauloid was investigated and compared with that of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39). Maximum activity of both enzymes is located in the growing region of the new phylloid. In the other parts of the frond, enzyme activity is only about one tenth of the values estimated for the intercalary meristem. The activity of PEP-carboxykinase: RuDP-carboxylase shifts from approxymately 9 in the growing region to only 3 in the old phylloid.

Correlations between photosynthetic enzymes, CO2-fixation and plastid structure in an albino mutant of Zea mays L.

SummaryAn albino seedling of Zea mays L. was investigated for its potential for CO2-assimilation. In the mesophyll the number, dimensions and fine structure of chloroplasts are drastically reduced but to a lesser extent in the bundle sheath. Chlorophyll concentration is zero and carotenoid concentration almost zero. Albinism also exerts a strong influence on the stroma of bundle sheath chloroplasts; ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) activity and glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) activity is not detectable. The C4-enzymes phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (decarboxylating) (EC 1.1.1.40) and the non-photosynthetic linked enzymes malate dehydrogenase (NAD) (EC 1.1.1.37), aspartate-2-oxoglutarate aminotransferase (EC 1.1.1.37), aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1.) and glyceraldehyde-3-phosphate dehydrogenase (NAD) (EC 1.2.1.1.) are present in the albino seedling with activities comparable to those in etiolated maize seedlings. The potential for CO2 fixation of the albino seedlings exceeds that of comparable dark seedlings considerably. The results are discussed with regard to enzyme localization of the C4 pathway of photosynthesis.

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

SummaryP730, 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.

[The control by phytochrome of phenylalanine ammonia-lyase activity in mustard seedlings (Sinapis alba L.)].

SummaryThe 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 Pfr (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 Pfr in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of Pfr 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 Pfr-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.

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

SummaryActinomycin D (10 μg/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).

Phosphoenolpyruvat-Carboxykinase und Ribulose-1,5-Diphosphat-Carboxylase von Laminaria hyperborea (Gunn.) Fosl.: Das Verteilungsmuster der Enzymaktivitten im Thallus@@@The distribution pattern of phosphoenolpyruvate-carboxykinase and ribulose-1,5-diphosphate carboxylase activities in fronds of Laminaria hyperborea (Gunn.) Fosl.

High activities of phosphoenolpyruvate-carboxykinase (EC 4.1.1.32) have been found in fronds of the brown alga Laminaria hyperborea (Gunn.) Fosl. The distribution pattern of this enzyme in the newly formed and in the old phylloid (last years production) and in the cauloid was investigated and compared with that of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39). Maximum activity of both enzymes is located in the growing region of the new phylloid. In the other parts of the frond, enzyme activity is only about one tenth of the values estimated for the intercalary meristem. The activity of PEP-carboxykinase: RuDP-carboxylase shifts from approxymately 9 in the growing region to only 3 in the old phylloid.SummaryHigh activities of phosphoenolpyruvate-carboxykinase (EC 4.1.1.32) have been found in fronds of the brown alga Laminaria hyperborea (Gunn.) Fosl. The distribution pattern of this enzyme in the newly formed and in the old phylloid (last years production) and in the cauloid was investigated and compared with that of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39). Maximum activity of both enzymes is located in the growing region of the new phylloid. In the other parts of the frond, enzyme activity is only about one tenth of the values estimated for the intercalary meristem. The activity of PEP-carboxykinase: RuDP-carboxylase shifts from approxymately 9 in the growing region to only 3 in the old phylloid.