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Porphyrin synthesis can be stimulated by a 16 h application of 5-aminolevulinic acid in combination with the chelator 2,2′-dipyridyl (DP), and reaches up to the 136-fold amount found in tissues of different organs of 5 or 7 day-old etiolated pea and maize seedlings, especially in their apical root segments. In all cases the dominating porphyrin is protoporphyrin IX (Proto), characterized by the quotient Proto/Mg-protoporphyrin (monomethylester) + protochlorophyllide amounting to 7 in root segments of Pisma sativum and up to 142 in root segments of Zea mays . The DP-induced synthesis of Proto in roots can be completely inhibited by gabaculine, an inhibitor of glutamate-1-semialdehyde-aminotransferase, a typical enzyme of the C 5 pathway. It can be concluded that the porphyrin synthesis up to protoporphyrinogen (Protogen) or Proto is located in the plastids of the roots as well as of the leaves. An export of Protogen or Proto from plastids into the cytosol takes place to a high degree in the roots (38% of the sum of porphyrins in all fractions), and to a very small degree in etiolated leaves (5% of the sum of porphyrins in all fractions), which means that it depends on the amount of the accumulated porphyrins in the organ. The absolute amount of Proto in root segments of different plant species does not correlate with the degree of photodestructions: the much lower stability of Proto in roots of Pisum sativum in comparison to Zea mays is discussed as consequence of the object-specific capacity to detoxify reactive oxygen species. The first degradation product of Proto in vivo and in vitro is the isomer photoprotoporphyrin.

The role of extra-porphyrin production in the magnesium branch of the porphyrin pathway was investigated in green pea ( Pisum sativum L.) leaves treated with 5-aminolevulinic acid (ALA) and 2,2′-dipyridyl (DP). The magnesium chelatase activity was estimated during protoporphyrin IX, Mg-protoporphyrin IX (monomethyl ester) (MgProto(E)) and protochlorophyllide accumulation in the presence of exogenous ALA and/or DP. The results showed a close correlation only between the enzyme activity and MgProto(E) levels. The inverse dependence of the magnesium chelatase activity on the amount of MgProto(E) accumulated supports the hypothesis that a mechanism is involved which controls the magnesium branch of chlorophyll biosynthesis in vivo through inhibition of the enzymes by their products. Illumination of intact pea leaves predarkened for 17 h resulted in a 1–2 h lag phase of magnesium chelatase activity with consequent strong stimulation. After 3 h of illumination it reached 200% compared with the enzyme activity immediately before illumination. Cycloheximide, if applied during the entire period of dark or light treatment, decreased the magnesium chelatase activity by 19% in the dark and by 39% in light, in comparison with the corresponding control variant on H 2 O. The ADP and especially the ATP content increased in the leaves treated with cycloheximide both in darkness and in light. The photosynthetic activity, measured as O 2 evolution by leaf segments, did not change in the presence of cycloheximide. The results are discussed as an additional influence of light on the magnesium chelatase activity not only via photosynthetic supply with ATP but also through light induced synthesis of the enzyme molecules de novo.

To obtain a clearer understanding of the photosensitization process, we have investigated the effect of photosensitization on the photochemical and non-photochemical energy dissipation in green leaves of wheat ( Triticum aestivum L.) and lettuce ( Lactuca sativa L.), pretreated with 5-aminolaevulinic acid (ALA) for 2–24 h in the dark, using chlorophyll (Chl) fluorescence quenching analysis and measurement of the influence on CO 2 uptake and xanthophyll cycle pigments. In response to dark pretreatment, leaves accumulated high levels of protochlorophyllide (PChlide) and non-metabolized ALA. The dark pretreatment had no effect on the intrinsic photochemical efficiency of photosystem II (PS II). Although quantitative differences exist between wheat and lettuce, exposure to actinic light caused significant effects with a similar overall response pattern in both plant species. Changes in excitation energy dissipation were obtained already by a very low photon flux density of 85 μmol photons m −2 s −1 within a few minutes CO 2 uptake was almost completely suppressed in photosensitized leaves, but they were able to build up the ΔpH necessary to drive de-epoxidation of violaxanthin. Fluorescence quenching analysis revealed that a progressive increase in non-radiative energy dissipation (measured as the non-photochemical quenching of Chl fluorescence, q N ) was paralled by a stronger reduction in the primary quinone electron acceptor of PS II (Q A ) with increasing time of ALA pretreatment in the dark. In the early stages of photosensitization, high-energy state quenching mainly contributed to q N . In the later stages, q N was dominated by a photoinhibitory component together with the photodegradation of xanthophyll cycle pigments, in particular antheraxanthin and zeaxanthin. The latter phenomenon appeared to be promoted in response to a highly increased reduction state of Q A . If ALA was simultaneously applied to leaves with 4,6-dioxoheptanoic acid, which acts as a competitive inhibitor of the enzyme ALA dehydratase, photosensitization disappeared when PChlide synthesis was completely inhibited, but was enhanced when inhibition was incomplete. Electron paramagnetic resonance studies using the spin trapping technique revealed a specific ALA-mediated formation of hydroxyl and carbon-centred radicals in response to actinic light exposure of chloroplasts. On the basis of these findings, a possible role of ALA is proposed: ALA enhances the photosensitizing effect(s) triggered by PChlide.

Summary In a preceding paper (Hartel et al., this issue, pp. 230–236), light-induced damage of structural components of the photosynthetic apparatus has been demonstrated after pretreatment of green leaf segments of wheat and lettuce with 5-aminolevulinic acid (ALA) in darkness. In the present study, the time course of this process has been investigated by means of chlorophyll fluorescence and measurement of the lightinduced electron spin resonance signal I of P700 + . After exposure of leaves pretreated with ALA in darkness to continuous light, a decrease in delayed chlorophyll fluorescence intensity coincided with an increase in P700 + formation. This was observed in both plant species during the first two hours, indicating disturbance of the electron transfer between the two photosystems. Later a complete inhibition of photosystem-II-mediated electron transfer was found in lettuce. P700+ measurements reflected a severe structural disintegration of thylakoid membranes which increased with duration of light exposure and light intensity, and which appeared to be accelerated when photosystem II activity was lost. In contrast, photodynamic damage in wheat leaf segments did not increase after 2 h of illumination. The electron transfer inhibition was incomplete, and P700 + measurements revealed no indication of strong structural injury.

The barley line albostrians exhibits a severe block in chloroplast development as a result of a mutationally induced lack of plastid ribosomes. White leaves of this mutant contain undifferentiated plastids, possess only traces of chlorophyll (Chl), and are photosynthetically inactive. Chl deficiency, combined with a continuous heme requirement, should lead to drastic changes in the tetrapyrrole metabolism in white versus green leaves. We analyzed the extent to which the synthesis rate of the pathway and the porphyrin distribution toward the Chl- and heme-synthesizing bifurcation is altered in the white tissue of albostrians. Expression and activity of several distinctively regulated enzymes, such as glutamyl-tRNAglu reductase, glutamate 1-semialdehyde aminotransferase, Mg- and Fe-chelatase, and Chl synthetase, were altered in white mutant leaves in comparison to control leaves. A drastic loss in the rate-limiting formation of 5-aminolevulinate and in the Mg-chelatase and Mg-protoporphyrin IX methyltransferase activity, as well as an increase in Fe-chelatase activity, accounts for a decrease in the metabolic flux and the re-direction of metabolites. It is proposed that the tightly balanced control of activities in the pathway functions by different metabolic feedback loops and in response to developmental state and physiological requirements. This data supports the idea that the initial steps of Mg-porphyrin synthesis contribute to plastid-derived signaling toward the nucleus. The barley mutant albostrians proved to be a valuable system for studying regulation of tetrapyrrole biosynthesis and their involvement in the bi-directional communication between plastids and nucleus.

The amounts of total phytol and its chlorophyll-bound and free portions have been determined quantitatively at different developmental stages of primary leaves of wheat using thin-layer chromatography combined with a potassium permanganate reaction. During the greening of etiolated leaves a close correlation exists between chlorophyll and phytol contents. Phytol is always present in excess and only in a bound state. During induced leaf senescence phytol is essentially degraded more slowly than chlorophyll. The liberated phytol does not influence the hydrolytic activity of chlorophyllase.