A. Hager

Die Isolierung und quantitative Bestimmung der Carotinoide und Chlorophylle von Blättern, Algen und isolierten Chloroplasten mit Hilfe dünnschichtchromatographischer Methoden

Methods for a rapid quantitative determination of chlorophylls and carotenoids are decribed.The extraction of pigments was carried out with different kinds of plant material, such as algae, leaves and chloroplasts. The separation of the carotenoids from these extracts was succeeded by an adsorptionchromatographic process in which the thin-layer consists of anorganic adsorbents (CaCO3, MgO, Ca(OH)2). The basicity of the layer is further increased by the addition of KOH; thereby the chlorophylls are retained at the starting line and the overlapping of chlorophylls and xanthophylls on the chromatogram can be avoided.With this method even those carotenoids can be separated which differ only in the position of a double bond, as for instance α- and β-carotene, and lutein and zeaxanthin. Thus the separation of all the principal carotenoids on a single chromatogram is possible, for example from a Chlorella extract (in order of decreasing Rf-value): α-carotene, β-carotene, lutein-5,6-epoxide (traces), violaxathin, lutein, antheraxanthin, neoxanthin neo A, neoxanthin and zeaxanthin. Furthermore the chromatographic behaviour of the carotenoids ζ-carotene, γ-carotene, lycopene and rhodoxanthin, which are found only rarely, is described.The chlorophylls a and b are separated by a partition chromatographic process on the second thin-layer; this layer consists of silica gel mixed with ascorbic acid as an antioxidant. An apparatus for an equal spreading of defined quantities of the extract on the starting line and new methods for a rapid quantitative elution of the pigments from the adsorbent are described.The specific extinction coefficients E 1 cm (1 %) for antheraxanthin in ethanol and for α-carotene in chloroform, which were needed for the calculation of the pigment quantity were determined.

Lichtbedingte pH-Erniedrigung in einem Chloroplasten-Kompartiment als Ursache der enzymatischen Violaxanthin- —- Zeaxanthin-Umwandlung ; Beziehungen zur Photophosphorylierung

1. During illumination of isolated chloroplasts (Spinacea oleracea) with red or white light in the presence of AS an immediate de-epoxidation of viol, to zea. takes place as in the case of intact cells. Inhibition of this light induced formation of zea. in isolated chloroplasts by DCMU is overcome by the addition of PMS + AS (pseudocyclic electron transport). PMS or PMS+FMN (without AS; cyclic electron transport) catalyze a conversion of viol, to zea. during illumination which is no longer inhibited by DCMU. In this case light reaction II of photo synthesis is not necessary for the xanthophyll interconversion. It can be shown that the formation of zea. takes place under conditions under which photophosphorylation takes place. A connection between these two processes seems to exist since uncouplers (CCCP, DNP, Methylamine, NH^) can completely eliminate the light dependent formation of zea. in chloroplasts (at pH 7 or higher). On the other hand, addition of ATP to chloroplasts can also trigger the con version of viol, to zea. without illumination.

Ausbildung von Maxima im Absorptionsspektrum von Carotinoiden im Bereich um 370 nm; Folgen für die Interpretation bestimmter Wirkungsspektren

1. Most carotenoids show a 3-peak-absorption curve in the visible spectral region in polar solutions. The addition of a definite quantity of H2O to such solutions (ethanol, methanol, aceton, isopropanol) changes the absorption curve of these pigments in a characteristic manner. A new peak appears in the uv region of the spectrum (e.g.in the case of lutein at 370 nm); simultaneously the 3-peak fine structure of the visible spectrum diminishes and completely disappears after further addition of H2O. Such changes are observed especially in the case of lutein and zeaxanthin, but also in the case of neoxanthin, violaxanthin and lycopene (of the carotenoids analyzed). During thermic excitation (45° C) the uv-peak in the carotenoid spectrum disappears and the normal 3-peak curve is restored; upon cooling the uv-peak appears again. The variation of the carotenoid spectrum and the formation of a maximum in the uv-region are possibly caused by an aggregation of the pigment molecules with participation of H2O molecules. This formation of polymers obviously leads to an alteration in the distribution of electrons in the chromophore system of the carotenoid molecule and thereby to a change of the light absorption. 2. Water-soluble carotenoid complexes isolated from spinach chloroplasts show a strong light absorption in the uv-region and a one-peak absorption curve in the visible blue. After transfer of the complex to polar solutions a characteristic 3-peak carotenoid curve appears in the blue region of the spectrum; concomitantly the maximum in the uv disappears. That means that carotenoids which are bound to membranes or particles in the intact cell may have a 4-peak absorption curve similar to that of pigments which are dissolved in the water-containing alcohols mentioned above. It is conceivable that those carotenoids which do not form uv peaks in the dissolved state are able to do so under conditions under which carotenoids are bound to membranes or particles. 3. The similarity of some action spectra to certain 4-peak, carotenoid spectra is striking. This is true particularly for the action spectrum of the first positive curvature of Avena coleoptile (Fig. 10). On the basis of the described abilities of the carotenoids to form an absorption peak in the wave uv, the appearance of such a maximum in an action spectrum (in the region about 370 nm) can no longer be considered to be sufficient proof for the participation of a flavin as light-acceptor.Summary1.Most carotenoids show a 3-peak-absorption curve in the visible spectral region in polar solutions. The addition of a definite quantity of H2O to such solutions (ethanol, methanol, aceton, isopropanol) changes the absorption curve of these pigments in a characteristic manner. A new peak appears in the uv region of the spectrum (e.g.in the case of lutein at 370 nm); simultaneously the 3-peak fine structure of the visible spectrum diminishes and completely disappears after further addition of H2O. Such changes are observed especially in the case of lutein and zeaxanthin, but also in the case of neoxanthin, violaxanthin and lycopene (of the carotenoids analyzed).During thermic excitation (45° C) the uv-peak in the carotenoid spectrum disappears and the normal 3-peak curve is restored; upon cooling the uv-peak appears again. The variation of the carotenoid spectrum and the formation of a maximum in the uv-region are possibly caused by an aggregation of the pigment molecules with participation of H2O molecules. This formation of polymers obviously leads to an alteration in the distribution of electrons in the chromophore system of the carotenoid molecule and thereby to a change of the light absorption.2.Water-soluble carotenoid complexes isolated from spinach chloroplasts show a strong light absorption in the uv-region and a one-peak absorption curve in the visible blue. After transfer of the complex to polar solutions a characteristic 3-peak carotenoid curve appears in the blue region of the spectrum; concomitantly the maximum in the uv disappears. That means that carotenoids which are bound to membranes or particles in the intact cell may have a 4-peak absorption curve similar to that of pigments which are dissolved in the water-containing alcohols mentioned above.It is conceivable that those carotenoids which do not form uv peaks in the dissolved state are able to do so under conditions under which carotenoids are bound to membranes or particles.3.The similarity of some action spectra to certain 4-peak, carotenoid spectra is striking. This is true particularly for the action spectrum of the first positive curvature of Avena coleoptile (Fig. 10). On the basis of the described abilities of the carotenoids to form an absorption peak in the wave uv, the appearance of such a maximum in an action spectrum (in the region about 370 nm) can no longer be considered to be sufficient proof for the participation of a flavin as light-acceptor.

Untersuchungen über die lichtinduzierten reversiblen xanthophyllumwandlungen an Chlorella und Spinacia

Summary1.Using new methods in thin-layer chromatography, experiments were carried out to prove the light-induced changes in the quantity of various xanthophylls in Chlorella and spinach leaves. The probable connection of these interconversions to electron transport in photosynthesis was demonstrated.2.The kinetics of these xanthophyll conversions were investigated during strong illumination and in the succeeding dark period (Chlorella).Already after illumination of 1 min one can detect a decrease of the di-epoxide xanthophyll violaxanthin and a corresponding increase of the epoxide-free zeaxanthin. The intermediate of this interconversion is the mono-epoxide antheraxanthin. Neoxanthin exhibits no change in concentration under the given light intensity and an illumination time of 60 min and more; the same result can be observed with the other carotenoids (α-carotene, β-carotene, lutein, lutein-5,6-epoxyd) and the chlorophylls a and b.3.The light-induced formation of zeaxanthin is not correlated with those pigment interconversions which are photooxidative in their nature and which may be detected only after long illuminations. However, by using damaged, e.g., briefly heated Chlorella cells, a photooxidative-induced decrease of carotenes and chlorophyll a and a smaller decrease of xanthophylls and chlorophyll b could already be demonstrated after illumination of 15 min. In this case the ratio xanthophylls/ carotenes increases.4.The transformation violaxanthin → antheraxanthin → zeaxanthin (“forward-reaction”) is induced not only by an illumination with white light (point 2) but also with red light (>600 nm); that means the reaction proceeds at a wavelength which cannot be absorbed by the xanthophylls themselves. Chlorophyll acts as light-acceptor.5.The “forward-reaction” does not proceed after the cells have been heated for a short time. The presence of inhibitors of light-reaction II in photosynthesis such as o-phenanthroline, hydroxylamine and DCMU entirely suppresses the above reaction. The inhibition by DCMU can be reversed by substances (in Chlorella) which initiate or increase the cyclic electron transport at chlorophyll aI: vitamin K5 and hexylresorcinol.In contrast to its effect in chloroplasts (unpublished results), salicylaldoxime is only a very weak inhibitor of xanthophyll-conversion. Cyanide does not influence the “forward-reaction”; furthermore the reaction can be observed under aerobic and anaerobic conditions. The light-induced formation of zeaxanthin is entirely suppressed by the uncouplers CCCP and methylamine in concentrations of 10-4 M and 5×10-4 M, respectively.6.The light-independent backward-reaction zeaxanthin → antheraxanthin → violaxanthin, which normally prevents a high accumulation of zeaxanthin, does not proceed under anaerobic conditions. Therefore under such conditions accumulation of zeaxanthin can be observed even in dim light.7.The results indicate that the light-induced transformation violaxanthin → antheraxanthin → zeaxanthin, which consists in the light-induced splitting of the epoxide oxygen from violaxanthin, is not identical with the process which cases the release of oxygen in photosynthesis. There is evidence, however, that the xanthophyll-conversion is coupled with that electron-transport which goes on between reduced plastoquinone and oxidized chlorophyll aI; energy-rich compounds which are formed in this step of electron transport or ATP itself apparently is responsible for the cleavage of the oxygen from violaxanthin and for the resulting formation of zeaxanthin.Zusammenfassung1.Mit Hilfe neuer dünnschichtchromatographischer Methoden wurden quantitative Untersuchungen über die lichtinduzierten Mengenänderungen der Xanthophylle in Chlorella und Spinatblättern durchgeführt und die möglichen Zusammenhänge dieser Mengenänderungen mit den Elektronentransport-Vorgängen bei der Photosynthese aufgezeigt.2.Der zeitliche Verlauf dieser Xanthophyll-Umwandlungen wurde während starker Belichtung und in nachfolgender Dunkelheit (Chlorella) untersucht.3.Die lichtinduzierte Violaxanthin → Antheraxanthin → Zeaxanthinumwandlung (”Hin-Reaktion“) findet auch im Rotlicht (>600nm) statt. Als Lichtacceptor fungiert das Chlorophyll.4.Diese Umwandlung hat nichts mit Pigmentveränderungen zu tun, die photooxydativer Natur sind, die erst nach längerer Belichtung in Erscheinung treten und die zu einer Erhöhung des Verhältnisses Xanthophylle/Carotine führen.5.Die Hin-Reaktion unterbleibt nach kurzzeitiger Erhitzung der Zellen. Sie wird ferner durch Hemmstoffe der Lichtreaktion II bei der Photosynthese, nämlich o-Phenanthrolin, Hydroxylamin und DCMU vollständig unterbunden. Die Hemmung durch. DCMU kann (in Chlorella) durch Substanzen wieder aufgehoben werden, welche einen cyclischen Elektronentransport am Chlorophyll aI hervorrufen oder verstärken: Vitamin K5 und Hexylresorcin.Im Unterschied zu Chloroplasten (unveröffentlicht) wird die Xanthophyllumwandlung durch den Kupfer-Komplexbildner Salicylaldoxim nur schwach gehemmt. Cyanid beeinflußt die Hin-Reaktion nicht. Sie läuft außerdem unter aeroben und anaeroben Bedingungen gleichermaßen ab.Die Entkoppler CCCP und Methylamin unterbinden die lichtbedingte Zeaxanthin-Bildung bei Konzentrationen von 10-4 bzw. 5×10-4 m bereits vollständig.6.Die lichtunabhängige Rückumwandlung Zeaxanthin → Antheraxanthin → Violaxanthin verhindert normalerweise eine stärkere lichtinduzierte Ansammlung von Zeaxanthin; diese Rückumwandlung wird in einer N2-Atmosphüre vollständig gehemmt. Unter solchen Bedingungen kann deshalb auch in schwachem Licht eine Akkumulation von Zeaxanthin beobachtet werden.7.Die Ergebnisse zeigen, daβ die lichtabhäangige Violaxanthin → Antheraxanthin → Zeaxanthin-Umwandlung indirekt an einen Transport von Elektronen gekoppelt ist, welcher zwischen dem lichtreduzierten Plastochinon und dem lichtoxydierten Chlorophyll a1 abläuft die bei diesem Elektronentransport gebildeten (energiereichen) Verbindungen oder das ATP selbst stehen in ursächlichem Zusammenhang mit der [O]-Abspaltung aus dem Violaxanthin und der daraus resultierenden Bildung von Zeaxanthin.

Veränderung der Lichtabsorption eines Carotinoids im Enzym (De-epoxidase)-Substrat(Violaxanthin)-Komplex

SummaryThe enzyme violaxanthin de-epoxidase catalysing the transformation of the xanthophyll violaxanthin to zeaxanthin has been isolated from spinach chloroplasts.Special properties of the enzyme make it possible for the carotenoid to be bound without initiation of any catalytic reaction; the isolation of an enzyme-substratecomplex is thereby greatly facilitated. After addition of cofactors to this complex the transformation of violaxanthin to zeaxanthin takes place.In this complex the light-absorption of violaxanthin is changed drastically: the normal three-peak absorption curve in the blue region of the spectrum is strongly decreased but in the uv-region around 380 nm a new absorption maximum appears.Recently a similar spectrum has been determined in vivo in the phototropic sensitive region of the sporangiophores of Phycomyces (Wolken, 1969) with the aid of microspectrophotometry.From these results it is concluded that part of the carotenoids occurring in plants is present in a protein-bound form and that these pigments show a considerably changed light absorption in comparison with the isolated pigment. The simultaneous occurrence of differently bound carotenoids may lead to the formation of 4-peak absorption curves (similar to those of flavines) with 3 maxima in the blue region and 1 maximum in the UV around 370–380 nm. These 4-peak curves are characteristic for many action spectra.It is emphasized that the strong absorption changes of carotenoids occurring during the binding of these pigments to proteins should be considered in analyzing difference spectra.

Zur Chromatographie der Lipoidlöslichen Blattfarbstoffe

ZusammenfassungI.Die Chloroplasten-Farbstoffe lassen sich zur quantitativen Bestimmung aus einem Aceton-Extrakt mit 10% iger NaCl-Lösung verlustlos in Berzin überführen und dann and einer präparierten Stärkesäule in die 6 Hauptfarbstoffe Neoxanthin, Chlorophyll b, Chlorophyll a, Violaxanthin, Lutein und Carotin aufteilen. Die Trennung wird durch ein Lösungsmittel-Gemische aus 5 Komponenten erreicht (in Volumeneinheiten: Benzin 60, Benzol 35, Chloroform 1,25, Aceton 0,55, Isopropanol 0,06).II.Eine neue papierchromatographische Methode gestattet es, unter besonderen Vorsichtsmaßregeln (Vakuum) die Blattpigmente nicht nur genauer in einzelne Farbstoff-Kompoenenten aufzuteilen, sondern sie auch in einem Analysengang in solchen Mengen zu gewinnen, daß sie spektroskopisch identifiziert und quantitativ erfaßt werden können. Mit dieser Methode wurden in höheren Pflanzen folgende Pigmente gefunden:a)Carotine: β-Carotin, β-Carotin-di-epoxyd, cfl, 13 Isomere; X0;b)Xanthophylle: Lutein, Violaxanthin, Neoxanthin, Zeaxanthin, Xanthophyllepoxyd, Chrysanthemaxanthin, Flavoxanthin, X1, X2, X3;c)Chlorophylle: Chloroophyll a, Chloropohyll b;d)Chlorophyll-Derivate: Phaeophytin a, Phaeophytin b, Phaeophorbid a, Phaeophorbid b, Chlorophyllide (a und b).

Untersuchungen üben die Rückreaktionen im Xanthophyll-Cyclus bei Chlorella, Spinacia und Taxus

SummaryThe epoxidation of zeaxanthin to the di-epoxide violaxanthin via the mono-epoxide antheraxanthin (called the backward-reaction), is examined with several plant objects and under various conditions.In Chlorella and in the needles of Taxus baccata a backward-conversion can be observed immediately after the termination of strong illumination. The reaction can be accelerated somewhat by exposure of the plant material to pure O2 or dim light.One cannot observe such an epoxidation in leaf disks of Spinacia oleracea under normal conditions (dark, air). It begins only under the influence of dim light or when pure oxygen is supplied. The absence of the backward-reaction under the given experimental conditions is a consequence of a closure of the stomata, which begins during the strong illumination and continues in the succeeding dark period; it is therefore a consequence of anaerobiosis in the plastid-containing cells. Yet a backward-reaction starts if the O2-tension in the cells is increased either by pure O2 given from outside or by the intracellular evolution of photosynthetic O2 (associated with a partial opening of the stomata).The concentration of the intermediate antheraxanthin increases strongly more at the beginning of the O2- or dim-light-promoted backward-reaction than the concentration of the endproduct violaxanthin. Hence it follows that during epoxidation the two O-atoms are added to the 5,6- and the 5′,6′-position of the zeaxanthin not simultaneously but one after the other.In isolated chloroplasts or cell fragments no backward-reaction could be observed under various conditions tested. An apparent backward-reaction in lyophilized cells or chloroplasts, which is triggered by light or O2-atmosphere, is a result of the different velocity of the photooxidative destruction of carotenoids.The in vivo epoxidation of zeaxanthin, which probably is catalysed by a Cu-containing enzyme, only proceeds in the presence of molecular O2.ZusammenfassungDie als „Rückreaktion” bezeichnete Epoxidierung des Zea. 2über das Monoepoxid Anth. zum Diepoxid Viol. wird an verschiedenen pflanzlichen Objekten und unter verschiedenen Bedingungen untersucht.In Chlorella und den Nadeln von Taxus baccata ist sofort nach Beendigung der starken Belichtung die Rückumwandlung zu beobachten. Diese ist durch Begasung des Pflanzenmaterials mit reinem O2 oder durch schwaches Licht noch etwas zu beschleunigen.In Blattscheibchen von Spinacia oleracea ist eine solche Epoxidierung unter Normalbedingungen (Dunkelheit, Luft) nicht festzustellen. Sie setzt erst unter dem Einfluß von schwachem Licht oder bei Zufuhr von O2 (100%) ein. Das Fehlen der Rückreaktion hat seine Ursache in einem unter den gegebenen Versuchsbedingungen stattfindenden Spaltöffnungsverschluß, der während der Starklichtgabe beginnt und während der nachfolgenden Dunkelheit noch anhält, und einer daraus resultierenden Anaerobiose in den plastidenhaltigen Zellen. Durch eine Erhöhung der O2-Spannung in diesen Zellen, entweder durch von außen zugeführten reinen O2 oder durch intracelluläre Entwicklung von Photosynthese-O2 (verbunden mit einer teilweisen Öffnung der Stomata), kommt es auch in den Spinatblättern zu einer Rückreaktion. Schwaches Licht kann also auf indirektem Weg die Rückreaktion beschleunigen.Die Konzentration des Zwischenproduktes Anth. nimmt bei der O2-oder schwachlichtgeförderten Rückreaktion anfänglich stärker zu als das Endprodukt Viol. Daraus folgt, daß bei der Epoxidierung die beiden O-Atome nicht gleichzeitig, sondern nacheinander in die 5,6- und 5′,6′-Stellung des Zea. eingebaut werden.In isolierten Chloroplasten oder Zellfragmenten konnte bisher unter verschiedensten Bedingungen keine Rückreaktion nachgewiesen werden. Eine in gefriergetrockneten Zellen oder Chloroplasten durch Licht und O2-Atmosphäre ausgelöste scheinbare Rückreaktion ist auf eine verschieden schnelle photooxidative Zerstörung der Carotinoide zurückzuführen.Die Epoxidierung des Zea., welche durch ein wahrscheinlich Cuhaltiges Enzym katalysiert wird, ist in vivo an das Vorhandensein von molekularem O2 gebunden.

Auxintransport und Phototropismus

SummaryShort illumination of excised coleoptiles (with or without apex) inhibits the subsequent transport of IAA-2-14C in these sections during darkness.To a certain extent the inhibition is dependent both on the light intensity and on the duration of illumination. Only the blue region of the visible spectrum is effective.The light induced inhibition is due to a decrease of the quantity of IAA transported; on the other hand, the velocity of transport remains unchanged.The inhibition of auxin transport can be observed only if coleoptiles contain endogenous or fed auxin during the preceding illumination period. Besides illumination inhibition of auxin transport can also be brought about by incubation of coleoptile sections with a previously illuminated IAA/FMN solution.Auxin transformed by peroxidase operates in the same way. The different oxidation products of IAA in the solutions used were identified: The only product which inhibits elongation growth and auxin transport was 3-M. The conversion of IAA to 3-M is accomplished by crude cell-free extracts from corn coleoptiles.An increased formation of labeled 3-M from IAA-2-14C during illumination of coleoptiles could be demonstrated.Since 3-M is not actively transported in coleoptiles, it must be assumed that 3-M functions as an inhibitor of auxin transport only at its site of formation.It is concluded that the phototropic curvature of coleoptiles and stems is triggered by the photooxidative formation of 3-M from IAA in the side exposed to light. The flow of growth substances will be partly blocked by 3-M in this side and can be directed to the shaded side.On the strength of these findings some phenomena of phototropism (transmission of stimulus, “mneme”, quantum yield) can easily be explained.