Untersuchungen über die Rhizosphäre

Ohne ZusammenfassungAusführlichere Darstellung in der gleichnamigen Dissertation, Mathematisch-Naturwissenschaftliche Fakultät der Universität Göttingen 1955.     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary In conidia-free submerged cultures of Neurospora crassa the various steps of light-dependend carotenoid synthesis were studied. The mycelium produces small amounts of pigments even in the dark. The data obtained are in part in good agreement with earlier results of Zalokar and show that the light-induced pigment production starts after a lag-period of 40 min and is finished after 6–8 hours; the photoreaction is saturated by relatively small dosages. In contrast to Zalokar's results we found that for photoinduction the reciprocity law holds true. The photoreaction is saturated by a certain amount of light independently of the light-intensity. Actidion (Cycloheximide) inhibits carotenoid synthesis completely when added before or up to 10 min after the onset of illumination, whereas addition 60 min after illumination already has no effect. Comparison with the results obtained with Fusarium shows that the reaction mechanism is very similar in both organisms, though the various steps seem to proceed faster in Neurospora.

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Herrn Prof. Dr. L. Brauner in Verehrung und Dankbarkeit zum 70. Geburtstag gewidmet.     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary Under anaerobic conditions Fusarium aquaeductuum is able to synthesize carotenogenic enzymes but does not produce pigments. If illumination of the mycelia in the presence of oxygen is followed by an incubation in the dark under N₂ atmosphere, the strictly concurrent formation of the different carotenoids sets off as soon as aerobic conditions are restored. The paraboloidal increase of pigment production possibly indicates that synthesis of carotenogenic enzymes is also resumed. Blocking this enzyme synthesis by addition of cycloheximide leads to a simultaneous and linear increase of each carotenoid portion as soon as oxygen is replenished. This is interpreted to mean that light induces carotenogenic enzymes in a coupled group. On the other hand, our present and earlier results do not support any hypothesis on the existence of a carotenogenic multienzyme complex. The composition of the pigment after carotenoid production has ceased provides evidence for a selective inhibition of the synthesis of individual carotenogenic enzymes. Changes in pigment composition caused by an incubation of the mycelia for 12 h under anaerobic conditions are also reported.     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary The purpose of these studies was to find which steps in the biosynthetic pathway of carotenoids in Fusarium are under photoregulation. After separation by column chromatography -carotene, neurosporene, -carotene, torulene, neurosporaxanthin and lycopene were identified from their absorption spectra in visible light and by co-chromatography tests with carotenoids from other organisms. No other carotenoids were detected. These components were each present in trace amounts (0.5–2 g/g dry weight) in stricly dark grown cultures. During incubation of the mycelium in buffered glucose solutions in darkness these carotenoids accumulated slowly but linearly with time.After a lag period of 30 min following photoinduction a sequential increase of the carotenoids occurs in the order mentioned above. With the exception of lycopene this sequence follows a decreasing degree of saturation. Addition of cycloheximide at various intervals during the incubation after illumination results in a differential inhibition of the synthesis of the various carotenoids, the pattern of which closely resembles the time sequence of increase of the carotenoids following photoinduction. It is therefore concluded that the synthesis of all these carotenogenic enzymes is photoinduced.Incubation of the mycelium with diphenylamine inhibits carotenoid synthesis; however, after illumination lower concentrations cause an increase in the amount of the more unsaturated components. Incubation with H₂O₂ results in the same time sequence of increase of the carotenoids as after photoinduction, whereas in incubations with mercuribenzoate the pattern is different. Synthesis of sterols (probably ergosterol) is not increased after illumination but is slightly decreased. From these results we conclude that the first step of the biosynthetic pathway on which photoregulation takes place lies between farnesyl-pyrophosphate and the coloured carotenoids.

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Die Untersuchungen wurden in großzügiger Weise von der Deutschen Forschungsgemeinschaft unterstützt.     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary The effect of dithionite, hydroxylamine and hydrogen peroxide on the light dependent carotenoid synthesis in Fusarium aquaeductuum has been investigated in order to find indications whether redox reactions are involved in the first steps of photoinduction.Addition of dithionite (5·10^-3 M/l) to the mycelium some time after illumination prevented carotenoid synthesis completely; however, when dithionite was removed after 30 min by washing the mycelium with buffer, Fusarium synthesized nearly the same amounts of carotenoids as it does without dithionite incubation. To prevent this direct effect on biosynthesis of pigments, the mycelium was treated for only 30 min at different times before and after a short illumination with buffered dithionite solution. When dithionite was present during the illumination or was applied up to 21/2 min after the lights had been switched off, no carotenoids were synthesized at all. The inhibitory effect of dithionite gradually decreased during a 171/2 min period following the end of the illumination time. After this period treatment with dithionite showed no irreversible influence whatsoever on the carotenoid synthesis. Essentially the same results were obtained when hydroxylamine (10^-2 M/l, freshly prepared) was used as a reducing agent.On the other hand incubation with buffered hydrogen peroxide solution (10^-2 to 10^-1 M/l) in the dark simulated the effect of illumination in inducing carotenoid synthesis. Both the kinetics of the pigment production and the inhibition by cycloheximide suggest that treatment with hydrogen peroxide in the dark truly substitutes for photoinduction. From these results it is concluded that dithionite and hydroxylamine are capable of reducing as yet unknown photooxidation products which are produced during illumination, as proposed by several authors. This oxidative action of light can be simulated by incubation of the mycelium with hydrogen peroxide.Furthermore results are presented which suggest that in Fusarium light acts in two ways: 1. it induces a de novo protein synthesis giving rise to an enhanced carotenoid production (light dependent synthesis) and 2. it inhibits a carotenoid synthesizing system (dark synthesis) which functions with low activity in the mycelium in the dark.     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Zusammenfassung Ziel der vorliegenden Untersuchungen was es, zu prüfen, ob der erste Schritt in der Reaktionskette der lichtabhängigen Carotinoidsynthese von heterotrophen Organismen, die photochemische Reaktion des Lichtacceptors, obligat sauerstoffbedürftig ist. Durch Belichtung unter anaeroben Bedingungen wird bei Fusarium aquaeductuum und Neurospora crassa Carotinoidsynthese induziert; die Farbstoffmenge im Mycel ist allerdings geringer als nach Belichtung unter aeroben Bedingungen. Bei beiden Pilzen wird in N₂-Atmosphäre Lichtsättigung bei etwa 5·10³ Lux·min erreicht und zwar unabhängig von der eingestrahlten Lichtintensität und Belichtungszeit; die höchste induzierbare Carotinoidmenge beträgt bei Fusarium 10%, bei Neurospora 75% von derjenigen, die nach Belichtung unter O₂ maximal gebildet wird. Führt man dem Mycel im Anschluß an eine sättigende Belichtung unter anaeroben Bedingungen im Dunkeln Sauerstoff zu, so wird durch eine darauf folgende 2. Belichtung eine zusätzliche Carotinoidsynthese induziert; der Photoreceptor wird also offenbar durch O₂ wieder reaktiviert. Diese Reaktivierung wird als Reoxidation des Photoreceptors (wahrscheinlich ein Flavin) interpretiert; sie ist innerhalb von 10 min abgeschlossen und temperaturunabhängig. Aus den Ergebnissen wird geschlossen, daß der Sauerstoff an der photochemischen Reaktion des Photoreceptors nicht direkt beteiligt ist, sondern nur als Elektronenacceptor für die Reoxidation des Photoreceptors dient.

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Light-dependent carotenoid SynthesisIV. The role of oxygen in photoinduction

Summary The purpose of these studies was to investigate wether the primary step in the reaction chain of the light-dependent carotenoid synthesis in heterotrophic organisms, the photochemical reaction of the photoreceptor, requires oxygen. Submerged cultures of Fusarium aquaeductuum and Neurospora crassa were used.Biosynthesis of carotenoids following a photoinduction occurred only under aerobic conditions. When the mycelia were illuminated in the absence of oxygen and subsequently transferred to aerobic conditions in the dark, they produced carotenoids; however, the amount of pigments produced was lower in comparison to that synthesized after photoinduction in O₂ atmosphere. Under anaerobic conditions light saturation of photoinduction occurred at a dosage of approximately 5.10³ Lux. min (white light) in both organisms; this dosage was independent of light intensity and time of illumination. The photoeffect followed the reciprocity law even in the absence of oxygen. The amount of pigments synthesized following a saturating photoinduction under anaerobic conditions was 10% in Fusarium and 75% in Neurospora of that after photoinduction under aerobic conditions. When mycelia were illuminated in N₂ atmosphere with saturating dosages, subsequently supplied with oxygen in the dark and then illuminated a second time with or without oxygen, an additional photoinduction took place. This result indicates that the photoreceptor can be reactivated by oxygen. This reactivation is completed in approximately 10 min; it is independent of temperature and does not occur in the absence of oxygen.From these results it is concluded that oxygen does not directly participate in the primary photochemical event but functions as an electron acceptor to keep the photoreceptor (possibly a flavin) in the proper oxidation state.

     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary The fungus Fusarium aquaeductuum synthesizes large amounts of carotenoids only after illumination or in the presence of mercuribenzoate (HMB) in the dark. The effect of HMB is abolished entirely by the addition of excess thiols such as cysteine or mercaptoethanol; evidently active HMB must be present continuously within the cell. In contrast, photoinduction needs only a short exposure to light to set off pigment production in a subsequent dark period. Illumination of mycelia preincubated with HMB in the dark induces an additional proportion of carotenoid synthesis which shows the same kinetics and quantitative yields as that observed in untreated controls. This demonstrates that HMB, though inhibiting O₂-uptake by 50%, does not interfere with light-induced carotenoid production as such. The effects of light and HMB are additive under various experimental conditions tested. After addition of thiols to mycelia treated with HMB in the light, carotenoid production decreased to an extent equal to the amount mediated by HMB in the dark controls. These results provide strong evidence that the mechanisms and the action sites of light and HMB in carotenoid synthesis are different. Incubation with HMB after exposure to light reduces the agent's effectiveness, which declines gradually to about 30% during the first two hours.These findings are interpreted to mean that in Fusarium there are two isoenzyme systems at work in carotenoid production: a constitutive one showing very low net activity is subject to direct stimulation by HMB; light somehow eliminates this enzyme system and, at the same time, induces an isoenzyme set which exhibits high carotenogenic activity and is insensitive to HMB treatment.

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Folgende Abkürzungen werden im Laufe dieser Arbeit benutzt HMB p-Hydroxymercuribenzoat - CMB p-Chloromercuribenzoat - CMS p-Chloromercuriphenylsulfonsäure     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary Anaerobic conditions completely block the synthesis of carotenoids in Fusarium aquaeduciuum or Neurospora crassa. Even after sufficient illumination of the mycelia in the presence of air, the subsequent production of carotenoids in the dark is suppressed entirely when the fungi are transferred to an oxygen-free atmosphere. In turn, restoring aerobic conditions sets off the pigment synthesis in the dark without renewed photoinduction at any time within 48 hours, although the yields of carotenoids decrease. Thus the photo-induced state appears to be fairly stable. In order to locate this state the sequence of light-induced reactions was specifically blocked before and after the synthesis of carotenogenic enzymes.Cycloheximide is a potent inhibitor of protein synthesis in fungi. When applied prior to illumination to either of the two molds it suppresses the photo-induced carotenoid formation, apparently by blocking the production of carotenogenic enzymes. This inhibition can be overcome by removing cycloheximide (e.g. by rinsing the mycelium with buffer) at any time (up to 30 h) during the dark period following illumination; however, gradually declining quantities of pigment are produced. These results provide evidence for the formation of a remarkably lasting induction product which can clearly be distinguished from the photooxidative product and from the carotenogenic enzymes.Under anaerobic conditions Fusarium is capable of producing plentiful amounts of carotenogenic enzymes, while Neurospora crassa forms only small quantities. In order to test the stability of these enzymes in vivo the following experimental setting was employed: in the presence of air the fungi were illuminated and subsequently kept in the dark for a period as long as the absolute lag phase of the pigment synthesis. Then cycloheximide was added to block any further protein synthesis, and at the same time the mycelia were transferred to a nitrogen atmosphere. Returning the fungi to aerobic conditions after various incubation periods resulted in a successively reduced pigment production. It thus appears that the activity of the photo-induced carotenogenic enzymes diminishes only slowly in vivo. These enzymes somehow seem to be stabilized as long as carotenogenesis is blocked.     

Studien über die UV-Mutabilität verschiedener Stämme von Serratia und über die Natur der Färbungsmutationen

Ohne ZusammenfassungHerrn Prof. Dr. Dr. h. c. B. Rajewsky zum 65. Geburtstag gewidmet.     

Untersuchungen über die Ernährungsphysiologie der Hausbockkäfer-Larven

Ohne Zusammenfassung     

Untersuchungen über die Regeneration von Farnprimärblättern

Ohne ZusammenfassungMit 17 Textabbildungen.     

Untersuchungen über die variabilität der Rädertiere

Ohne ZusammenfassungHerrn Prof. Dr. K. v. Frisch zum 75. Geburtstag in Dankbarkeit und Verehrung gewidmet.     

Untersuchungen über die Sexualität der Myxomyceten

Ohne ZusammenfassungMit 3 Textabbildungen und Tafel I.     

Versuche über die Keimfähigkeit alter Sämereien

Ohne Zusammenfassung     

Untersuchungen über die Bedeutung der Rhizosphäre

Zusammenfassung Die Rhizosphäre tritt bei weißem Senf (Sinapis alba) nicht nur in reinem Quarzsand, sondern auch in Wasserkultur deutlich in Erscheinung.Beliebig ausgewählte Reinkulturen von Mikroorganismen (sporenbildende Erdbakterien, rote Hefe) vermögen in einer sterilen Kultur von weißem Senf normale Rhizosphärenbilder zu erzeugen, so daß jedenfalls eine Spezifität der Rhizosphärenbesiedler noch nicht erwiesen erscheint.Sterile Pflanzen von weißem Senf bilden in sterilem Quarzsand erheblich weniger Kohlensäure als mit Rhizosphäre behaftete Pflanzen. Die Bodenatmung ist also nicht, wie Ehrenberg meint, in erster Linie der Tätigkeit der Pflanzenwurzeln zuzuschreiben.Diese Tatsache muß auch bei Stoffwechseluntersuchungen beachtet werden. Eine sichere Bilanz des Kohlenstoffumsatzes läßt sich nur an sterilen Pflanzenkulturen erzielen.Ein fördernder Einfluß von Mikroorganismen aus der Rhizosphäre auf die Keimung der Samen von weißem Senf konnte nicht festgestellt werden. Beobachtete Förderungen durch Zusatz von Erdauszügen erwiesen sich als Förderung durch erhöhte Acidität¹.