D. Dörnemann

The regulatory role of polyamines in structure and functioning of the photosynthetic apparatus during photoadaptation

In this contribution we describe the changes in structure and functioning of the photosynthetic apparatus of the unicellular green alga Scenedesmus obliquus induced by inhibition or induction of polyamine biosynthesis. The synthesis or inhibition is controlled by white light of low and high intensities, as well as by blue and red irradiation. We observe that a decrease of the intracellular putrescine level and, vice versa, an increase of spermine, indicated by a raised ratio of spermine to putrescine, simulate a low-light photoadapted photosynthetic apparatus. Action spectra of the spermine/putrescine ratio compared with action spectra on characteristics of photoadaptation, e.g., chlorophyll biosynthesis, or the rate of primary photochemistry and electron transport, show that the photoreceptors for both the adaptation of the photosynthetic apparatus to low-light conditions and for the regulation of the spermine/putrescine ratio are, at least, very similar. We show that the photoreceptor is primarily a blue-light receptor with a superimposed red-light receptor that absorbs around 680 nm.

Light-dependent induction of strongly increased microalgal growth by methanol

Low methanol concentrations (about 0.5% v/v) induce biomass production in cultures of the unicellular green alga Scenedesmus obliquus by more than 300%, compared to controls without this solvent. This effect on the microalgal growth was found to be dependent on the solvent concentration, the packed cell volume (PCV), light intensity and light quality. It could be shown that methanol addition leads to a decrease in size of the light harvesting complex (LHC) on the basis of chlorophylls and proteins, and thus to changes in structure and functioning of the photosynthetic apparatus. These alterations lead to enhanced photosynthesis and respiration rates. The action of methanol on the photosynthetic apparatus is thus comparable to the effect of enhanced CO(2) concentrations. These findings support the previously proposed pathway for methanol metabolization with CO(2) as the final product. We conclude that the subsequent assimilation of the increased CO(2) amounts by the Calvin-Benson cycle is a possible explanation for the methanol-mediated increase in biomass production in terms of PCV. The methanol effect is observed only in the light and in the presence of a functioning photosynthetic apparatus. Preliminary action spectra suggest that the primary photoreceptor is a chlorophyll-protein complex with two absorption maxima at 680 and 430 nm, which may possibly be attributed to the reaction center of photosystem II (PSII).

INFLUENCE OF THE HABITAT ALTITUDE ON THE (PROTO) HYPERICIN AND (PROTO)PSEUDOHYPERICIN LEVELS OF HYPERICUM PLANTS FROM CRETE

Environmental factors are known to influence strongly the accumulation of secondary metabolites in plant tissues. In a previous paper, we studied the contents of (pseudo)hypericin and its immediate precursors in wild populations of various HYPERICUM species on the island of Crete, Greece, in dependence on their developmental stage. In this study, we investigated the effect of the habitat altitude on the total hypericins content of the plants, which is defined as the sum of protohypericin, hypericin, protopseudohypericin and pseudohypericin. Taking into account our previous finding that the highest accumulation is found during the flowering period in June, we collected the aerial parts of spontaneously growing H. PERFORATUM L. , H. TRIQUENTRIFOLIUM Turra , H. EMPETRIFOLIUM Willd. and H. PERFOLIATUM L. during that time frame at elevations between 100 and 600 m above sea level, however, bearing in mind the time lag in development with increasing altitude. HPLC analysis of the plant material, separated again into a flowers and a leaves/petioles fraction, revealed great differences in the total hypericin content in dependence on the altitude of the habitat. Specifically, a clear trend was revealed, showing an increase of the total hypericin content with increasing altitude. However, no changes could be observed in the ratio of hypericin to protohypericin and in that of pseudohypericin to protopseudohypericin. The habitats of the employed plants were again randomly distributed all over Crete. It is proposed that higher light intensities accompanied by enhanced UV-B radiation and lower air temperature might be responsible for the increasing levels of total hypericins with increasing altitude

Quantitative determination of 4,5-dioxovaleric acid as a metabolite in Scenedesmus obliquus after complete separation from 5-aminolevulinic acid

Abstract The involvement of the C5-pathway in the biosynthesis of 5-aminolevulinate for the formation of chlorophyll is proven beyond doubt. At least two reaction mechanisms have been proposed for the C5-pathway, which converts glutamate to 5-aminolevulinate: one involves 4,5-dioxovalerate and the other glutamate-1-semialdehyde. Evidence for the intermediates in vivo is generally considered of great importance for the proof of one or the other pathway. However, another favourable role for 4,5-dioxovalerate is as an intermediate in the conversion of glutamate-1-semialdehyde to 5-aminolevulinate. A recent report that 4,5-dioxovalerate is only an artefact formed duriing the assay [1] contradicts earlier results identifying it in extracts from Scenedesmus cells [2,3]. In the present paper the in vivo formation of thic compound is again demonstrated by different methods of isolation and characterization, including TLC and HPLC followed by absorption and fluorescence measurements and mass spectroscopy. The objection that the diaminonaphthalene derivative of 4,5-dioxovalerate is formed exclusively by the condensation of 5-aminolevulinate with 2,3-diaminonaphthalene [1] is disproved in this paper and the formation and existence of 4,5-dioxovalerate in vivo is confirmed.

Characterization of the photoreceptor(s) responsible for the regulation of the intracellular polyamine level and the putative participation of heterotrimeric G-proteins in the signal transduction chain

Abstract In previous publications we have demonstrated a photoregulation of the intracellular polyamine content during chloroplast development. In the present paper action spectra of the changes in the intracellular polyamine levels of putrescine (Put), spermidine (Spd) and spermine (Spm) are presented, giving first evidence for the existence of three photoreceptor systems: a protochlorophyllide photoreceptor is possibly responsible for the inhibition of Put and Spd formation during chloroplast development; a blue-light photoreceptor that probably mediates in general the formation of polyamines, possibly in the semiquinone form in the case of Spm; and, finally, a red-light (possibly the PSII reaction centre) photoreceptor that could be responsible for the induction of the polyamine increase. The function and the physiological role of these three photoreceptor systems are discussed. Although chloroplast photodevelopment and the inhibition of polyamine biosynthesis have the same photoreceptor, it seems that this inhibition is not directly linked to the signal transduction chain of chlorophyll biosynthesis. However, there are hints that a separate transduction chain, in which heterotrimeric G-proteins are involved, leads to the inhibition of polyamine biosynthesis. Dark-grown cultures, supplemented with a non-hydrolysable GTP analogue, GTP-γ-S, as well as cells treated with cholera (CTX) and pertussis (PTX) toxins, simulate polyamine changes similar to those of cells grown under light qualities that do not significantly influence chlorophyll biosynthesis. A possible hypothesis for the mode of interaction of polyamine regulation and chloroplast development is presented.

The inhibitory effect of 4,5-dioxovalerate on 5-aminolevulinate dehydratase and its implication in the regulation of light-dependent chlorophyll formation in pigment mutant C-2A′ of Scenedesmus obliquus

Besides the known role of 4,5-dioxovalerate as an intermediate in the C 5 -pathway [1], a novel regulatory function in light-dependent chlorophyll biosynthesis is described. Considerable amounts of protochlorophyllide accumulate in dark grown cultures of the yellow mutant C-2A′ of Scenedesmus [2]. This accumulation is almost completely blocked in darkness by the addition of 4,5-dioxovalerate in vivo. Likewise, light-dependent chlorophyll biosynthesis is strongly inhibited by the addition of this compound during greening. The considerable increase of protochlorophyllide formation in darkness upon the addition of 5-aminolevulinate (Kotzabasis, K. and Senger, H. (1989) Z. Naturforsch., in press) is also drastically reduced by external 4,5-dioxovalerate. It is shown by in vitro experiments that concentrations of dioxovalerate, above the physiologically relevant level, inhibit 5-aminolevulinate dehydratase. The K i -value was determined to be 60±5 μM. From these results it is concluded that besides the predominant control of gli t-RNA-ligase by protochlorophyllide [4] a second regulatory mechanism is involved in chlorophyll biosynthesis. Under excessive concentrations of 5-aminolevulinate and 4,5-dioxovalerate further prophobilinogen and subsequently chlorophyll biosynthesis is inhibited.

The Regulatory Role of Polyamines on the Structural and Functional Photoadaptation of the Photosynthetic Apparatus

The adaptation of the photosynthetic apparatus to low and high light intensities is a well documented phenomenon, as well in higher plants [1], as in green algae [2]. The organisms respond to shade conditions by increased amounts of chlorophyll at a lowered photosynthetic capacity. Their photosynthetic compensation point is shifted to lower light intensities and the respiratory oxygen uptake decreased. Low light intensities can be mimiced by monochromatic red light in higher plants [3] and by blue light in algae [4]. The photoreceptors responsible for the adaptation to the different light qualities are phytochrome [5] and blue light photoreceptors [6], respectively. Changes in the molecular organization of the photosynthetic apparatus of green algae adapting to low intensities of white light result in an increase of the light harvesting complex, preferentially of photosystem II [7]. The ability of green algae to adapt unexpectedly fast (in about 8h, [8]) to new irradiation conditions demonstrates the high capability of the photosynthetic apparatus to respond to changes in the environmental conditions.

Intermediates, Catalytic Components and Light and Dark Regulation of ALA and Chlorophyll Formation in the Green Alga Scenedesmus

The nature of the intermediates of the C5-pathway has been a matter of controversy for a long time. Although the involvement of glutamate-1-semialdehyde (G-l-SA) (1) is widely accepted, the role of 4,5-dioxovalerate (DOVA) (2) as a catalytic component in the C5-pathway is still challenged (3). Well established is yet the activation of glutamate for the formation of 5-aminolevulinate (ALA) by a tRNAglu as demonstrated for barley (4), Chlamydomonas (5), Chlorella (6), Methanobacterium (7), Euglena (8), Cyanidium (9), Synechocystis (9) and spinach (9).

EFFECT OF LIGHT QUALITY ON THE in vitro THYLAKOID PROTEIN PHOSPHORYLATION IN THE GREEN ALGA Scenedesmus obliquus

Abstract Thylakoid protein phosphorylation was assayed in vitro with isolated thylakoids of Scenedesmus obliquus by irradiation with monochromatic light of different wavelengths and equal photon fluences. The action spectrum for light‐activated protein phosphorylation showed two maxima at 450 and 679 nm. A minimum of activity was reached around 580 nm. At this wavelength, the level of protein phosphorylation barely differed from that of dark‐incubated samples. The action spectrum of thylakoid protein phosphorylation resembled the chlorophyll absorption spectrum obtained in vivo. The results show that chlorophyll is the photoreceptor for thylakoid membrane phosphorylation.

Regulatory Effects of Polyamines on Chloroplast Development

The main naturally occuring polyamines, the diamine putrescine (Put), the triamine spermidine (Spd) and the tetramine spermine (Spm) are ubiquitous in living organisms [1]. At cellular pH values, polyamines behave as polycations, and can interact with anionic macromolecules such as DNA, RNA, phospholipids and certain proteins [1]. This association of polyamines with macromolecules is generally thought to constitute the physical basis for their physiological action. The possibility that polyamines could play a role in photosynthesis was suggested by their occurrence in isolated chloroplasts [2] and isolated chlorophyll/protein complexes [3]. Besford et al. [4] identified D1, D2, Cyt f and the large Rubisco subunit as the proteins which were stabilized by the addition of exogenous polyamines. Andreadakis and Kotzabasis [2] as well as Dornemann et al. [5] showed that the level of prolamellar body/prothylakoid-bound polyamines is higher than the corresponding one in thylakoids.