Edgar Wagner

Oxidative Stress Induces Partial Degradation of the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Isolated Chloroplasts of Barley.

The effects of oxidative stress on the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were studied in isolated chloroplasts from barley (Hordeum vulgare L. cv Angora). Active oxygen (AO) was generated by varying the light intensity, the oxygen concentration, or the addition of herbicides or ADP-FeCl3-ascorbate to the medium. Oxidative treatments stimulated association of Rubisco with the insoluble fraction of chloroplasts and partial proteolysis of the large subunit (LSU). The most prominent degradation product of the LSU of Rubisco showed an apparent molecular mass of 36 kD. The data suggest that an increase in the amount of AO photogenerated by O2 reduction at photosystem I triggers Rubisco degradation. A possible relationship between AO-mediated denaturation of Rubisco and proteolysis of the LSU is discussed.

Plasma Membrane H + -ATPase Is Involved in Auxin-Mediated Cell Elongation during Wheat Embryo Development

Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H+-ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two–three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H+-ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H+-ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H+-ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.

Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis

Abstract. Active oxygen (AO) species generated in plants under stress conditions trigger degradation of Rubisco (EC 4.1.1.39). To find out whether AO species activate proteases or make the protein susceptible to proteolysis, purified and 14C-labelled Rubisco protein was incubated with stromal preparations obtained from barley (Hordeum vulgare L.) leaves. The protein was degraded into distinct fragments only after a treatment with AO. This result shows that AO-treated Rubisco has been modified to become a substrate for stromal protease(s) and dismisses the possibility of protease activation. Upon degradation, distinct fragments accumulated with time. The fragmentation pattern was indistinguishable from that obtained with intact chloroplasts subjected to oxidative conditions (cf. M. Desimone et al., 1996, Plant Physiol 111: 789–796). Degradation required ATP-hydrolysis, since AMP, ADP or non-hydrolysable ATP-analogs did not support proteolysis. The ClpP-deficient stromal preparations degraded AO-modified Rubisco, making the involvement of the ClpC/P protease unlikely.

An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast

Glutathione S-transferases (GSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max L.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic GST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic GST but did not affect the intracellular isoforms. The discovery of apoplastic inducible GST will be discussed in light of the putative function of these enzymes in plants.

Endogenous rhythmicity and energy transduction : IV. Rhythmic control of enzymes involved in the tricarboxylic-acid cycle and the oxidative pentose-phosphate pathway in Chenopodium rubrum L.

Summary1.All enzymes tested: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G-6-PDH), and 6-phosphogluconate dehydrogenase (6-P-GDH)_behaved rhythmically during a period of continuous white light interrupted by darkness. All showed approximately the same frequency of 12–15 h.2.The enzymes of the tricarboxylic-acid cycle (MDH and GDH) are in phase with each other but are out of phase with those of the oxidative pentose-phosphate pathway (G-6-PDH and 6-P-GDH) which are in turn in phase with each other.3.GDH appears to be activated by the addition of Hoaglands solution which leads to an overt rhythm 24 h prior to darkness. The rhythms of MDH, G-6-PDH and 6-P-GDH cannot be demonstrated prior to the onset of darkness due to an inhibition of the MDH and pentose-phosphate cycle enzymes by light.4.The control of the frequency and phase of these rhythms are discussed in relation to a positive correlation of the rhythms in enzyme activity presented here and the rhythms of the pyridine nucleotides presented elsewhere.

“Primäre” und “Sekundäre” Differenzierung im Zusammenhang mit der Photomorphogenese von Keimpflanzen (Sinapis alba L.)

SummaryAnthocyanin synthesis of the mustard seedling (Sinapis alba L.), a typical phytochrome-dependent photoresponse has been further investigated. — It has been found that only two types of tissue can synthesize anthocyanin under the influence of active phytochrome (=P730), namely, the epidermis of the votyledons and the subepidermal layer in the hypocotyl (Fig. 2, 3). — Under our standard conditions (25° C; cf. methods) phytochrome-potentiated anthocyanin synthesis is only possible 24 hours after sowing and it ceases about 60 hours after sowing, independent of the amount of anthocyanin which has been accumulated (Fig. 5, 6). On the basis of the whole seedling the highest sensitivity of the anthocyanin producing system to light is around 36 hours after sowing (Fig. 8). Within the tissues which are capable of forming anthocyanin there is a characteristic shift of the ability to respond to P730 as the seedling ages. If we devide the seedling into 4 segments (Fig. 9) it turns out that in the basal and middle part of the hypocotyl the ability to form anthocyanin is rapidly lost whereas in the upper part of the hypocotyl and in the cotyledons this ability even increases at first. The following decrease is slower than in the basal parts (Fig. 10, 11).It is argued that this specific and dynamic cellular pattern of responsiveness to P730 can be regarded as a manifestation of a “primary differentiation” in the course of which the genotype of each individual cell in the dark-grownt seedling is devided into 3 functional types of genes: active, inactive, and potentially active genes (P730) (Fig. 4). — In connection with anthocyanin synthesis P730 is thought to act exclusively at the level of “secondary differentiation”, i.e., it is thought to initiate the action of potentially active genes via a signal-chain. The action of P730 is non-specific. The specificity of the photoresponse of an individual cell is determined by the status of its “primary” differentiation (Fig. 4).If the process of differentiation is slowed down (e.g. by the application of low doses of Actiomycin D) anthocaynin synthesis can continue much longer than under our standard conditions where it ceases around 60 hours after sowing (Fig. 12). This fact seems to indicate that the loss of the ability to form anthocyanin is due to an inactivation of pertinent genes by the process of “primary differentiation”, which is itself, as one would expect, under the control of genes.ZusammenfassungDie Anthocynsyanthese des Senfkeimlings ist phytochromabhängig. Lediglich zwei Gewebe, die Epidermis der Cotyledonen und die Subepidermis des Hypocotyls sind zu dieser Anthocyansynthese fähig. Erst 24 Std nach Aussaat ist Anthocyansynthese möglich und bereits etwa 60 Std nach Aussaat (25° C; Standardbedingungen vgl. Methoden) erlischt dei Fähigkeit zur Anthocyansynthese weitgehend und zwar unabhängig von der Menge des synthetisierten Anthocyans. Die höchste “Empfindlichkeit” für Licht besitzt das Anthocyan bildende System etwa 36 Std nach Aussaat. — Teilt man den Keimling unmittelbar vor der Anthocyanmessung in 4 Segmente auf (Abb. 9) und mißt den Anthocyangehalt der Segmente getrennt, so stellt sich heraus, daß die Fähigkeit zur Anthocyansynthese im mittleren und basalen Bereich des Hypocotyls rapide verloren geht. Im oberen Hypocotylabschnitt hingegen und in den Cotyledonen nimmt diese Fähigkeit erst zu, und die Abnahme ist langsamer. —Es werden Argumente für die Auffassung entwickelt, daß das spezifische und dynamische Zellmuster, das man hinsichtlich der P730-abhängigen Anthocyansynthese vorfindet, ein Ausdruck der “primären” Differenzeierung sei (vgl. Abb. 4). P730 hingegen, so stellen wir uns vor, wirkt unspezifisch im Rahmen einer “sekundären” Differenzierung, indem es “potentiell aktive Gene (P730)” in Funktion setzt. Welche Gene in den einzelnen Zellen des Dunkelkeimlings potentiell aktiv sind, legt die primäre Differenzierung fest. — Diese Vorstellungen werden durch den Befund gestützt, daß eine Applikation von Actinomycin D zu einer zeitlich sehr viel ausgedehnteren Anthocyansynthese führt; offenbar deshalb, weil die genetisch kontrollierte Entwicklung des primären Differenzie-rungsmusters gebremst wird. Eine Folge wäre, daß die “Inaktivierung” der zur Anthocyansynthese benötigten Gene, die normalerweise etwa 60 Std nach Aussaat erfolgt, weit hinausgezögert wird.

Epidermal growth factor receptor and transforming growth factor alpha expression in human ovarian carcinomas.

The varying tumorbiological behaviour of ovarian carcinomas probably influences operability, response to chemotherapy, being one of the most relevant prognostic factors. Because it is believed that an activation of the epidermal growth factor/transforming growth factor alpha (EGF/TGF alpha) signal pathway could be involved, we analysed the expression of epidermal growth factor receptor (EGFR) and TGF alpha with molecular-chemical, biochemical and immunohistochemical methods in 42 ovarian carcinomas, 4 ovarian metastasis, 2 other malignant ovarian tumours, and in 25 nonmalignant tissues (ovary, myometrium). No major rearrangements or amplification of the EGFR or TGF alpha genes were found. In non-malignant tissues no strong EGFR or TGF alpha signals were detected. TGF alpha is mainly produced by the tumour cells as shown by immunohistochemistry. Four different high molecular weight forms (20-48 kD) were detected in malignant tissues by western blot analysis.

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings.

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.

Pyridine nucleotides and redox-charge evolution during the induction of flowering in spinach leaves

In the long-day plant Spinacia oleracea changes in the pool size of pyridine nucleotides have been followed under different photoperiodic conditions. In short days (vegetative state), the dark and light phases of the cycle are characterized by specific reciprocal changes in NAD and NADP pool sizes. As a consequence, the ratios of NADH/NAD+NADH and NADPH/NADP+NADPH, which are respectively considered to represent the catabolic and anabolic state of metabolism, also show a characteristic pattern. Upon transfer to continuous light, i.e. during floral induction, a decrease in anabolic metabolism is paralleled by an increase in catabolic metabolism. In the floral state, both the catabolic and the anabolic couples of the pyridine nucleotides are considerably depressed, possibly reflecting the enhanced senescence of induced leaves. The results are discussed in relation to the involvment of the nucleotides in stoichiometric coupling of metabolic compartments at the cellular level in response to environmental signals.

Kinetische studien zur Interpretation der Wirkung von Sukzedanbestrahlungen mit Hellrot und Dunkelrot bei der Photomorphogenese (Anthocyansynthese bei Sinapis alba L.)

SummaryIn a previous paper (Bertsch and Mohr, 1965) we reported that in light-induced anthocyanin synthesis of the mustard seedling (Sinapis alba L.) a preirradiation with far-red light increases the effectiveness of a following irradiation with red light, whereas a preirradiation with red reduces the effectiveness of a following irradiation with far-red (Table 1). The amount of anthocyanin present 48 hours after the onset of the irradiation programme was taken as a gauge for the effectiveness of the irradiation with succedaneous red and far-red (and vice versa).In the present paper it is shown—using detailed kinetical studies (Fig. 1 and 2) —that a specific potentiating effect of the preceding far-red is not involved. The apparent effect is due to the fact that the preceding far-red eliminates the lag-phase for the following red (Fig. 1). — On the other hand, the depressing effect of red light preceding far-red is very real. This latter effect must be attributed to a loss of phytochrome.We demonstrate in the present paper that the effects of succedaneous red and far-red irradiations can be attributed altogether to phytochrome if several assumptions concerning the stability of phytochrome 730 (Hartmann, 1966; Wagner and Mohr, 1966) are made. These assumptions seem to be well justified. — In any case our kinetical studies have revealed no data which indicate that in red or far-red light we have to deal with anything else except phytochrome.ZusammenfassungIn einer früheren Arbeit (Bertsch und Mohr, 1965) haben wir bei der lichtinduzierten Anthocyansynthese des Senfkeimlings gefunden, daß eine Vorbestrahlung mit Dunkelrot die Wirkung einer nachfolgenden Bestrahlung mit Hellrot steigert. Eine Vorbestrahlung mit Hellrot hingegen reduziert die Wirksamkeit einer nachfolgenden Bestrahlung mit Dunkelrot (Tabelle 1). Die 48 St nach Beginn des Bestrahlungsprogramms vorhandene Menge an Anthocyan wurde als ein Maß für die Wirksamkeit der Sukzedanbestrahlungen angesehen. — In der vorliegenden Arbeit wurde mit Hilfe kinetischer Studien gezeigt, daß ein spezifischer Effekt der Dunkelrot-Vorbestrahlung nicht existiert. Der apparente Effekt ist darauf zurückzuführen, daß das zuerst gegebene Dunkelrot die lag-Phase für das nachfolgende Hellrot eliminiert. — Der Effekt, daß eine Hellrot-Vorbestrahlung die Wirkung von nachfolgendem Dunkelrot stark reduziert, ist hingegen real. Dieser Effekt muß auf einen Verlust an Phytochrom zurückgeführt werden.In a previous paper (BERTSCH and MOHR, 1965) we reported that in light-induced anthocyanin synthesis of the mustard seedling (Sinapis alba L.) a preirradiation with far-red light increases the effectiveness of a following irradiation with red light, whereas a preirradiation with red reduces the effectiveness of a following irradiation with far-red (Table 1). The amount of anthocyanin present 48 hours after the onset of the irradiation programme was taken as a gauge for the effectiveness of the irradiation with succedaneous red and far-red (and vice versa).In the present paper it is shown-using detailed kinetical studies (Fig. 1 and 2) -that a specific potentiating effect of the preceding far-red is not involved. The apparent effect is due to the fact that the preceding far-red eliminates the lag-phase for the following red (Fig. 1). - On the other hand, the depressing effect of red light preceding far-red is very real. This latter effect must be attributed to a loss of phytochrome.We demonstrate in the present paper that the effects of succedaneous red and far-red irradiations can be attributed altogether to phytochrome if several assumptions concerning the stability of phytochrome 730 (HARTMANN, 1966; WAGNER and MOHR, 1966) are made. These assumptions seem to be well justified. - In any case our kinetical studies have revealed no data which indicate that in red or far-red light we have to deal with anything else except phytochrome.