Comparative analysis of the low molecular weight and enzymatic antioxidants in response to the phototoxicity of accumulating uroporphyrin and protochlorophyllide in barley leaves treated with cesium chloride.

Cesium chloride treatment of illuminated barley leaves leads to accumulation of uroporphyrinogen which is subsequently either oxidised to uroporphyrin in continuous light or converted to protochlorophyllide in darkness [Shalygo et al. (1998) J Photochem Photobiol 42: 151-158]. We were interested to elucidate the differences in the phototoxicity of uroporphyrin and protochlorophyllide in the CsCI-treated leaves. Photosensitization and the induction of oxidative stress responses in the barley leaves occurred much faster upon protochlorophyllide than upon uroporphyrin accumulation. We compared the time resolved changes in the pool sizes of low molecular weight antioxidants, such as ascorbate, glutathione and tocopherol, as well as of the enzymatic activities of catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase in illuminated barley leaves which accumulated uroporphyrin or protochlorophyllide. A rapid loss of the antioxidant levels correlated with the accumulation of reactive oxygen species. The contents of low molecular weight antioxidants and the activities of most of the antioxidative enzymes declined more rapidly in the presence of protochlorophyllide than of uroporphyrin. Due to its high lipophilicity, free protochlorophyllide is associated with biomembranes. Therefore, it is assumed that it exerts its phototoxic effects to membranes more rapidly than uroporphyrin.

Photodynamic action of uroporphyrin and protochlorophyllide in greening barley leaves treated with cesium chloride.

Incubation of greening barley leaves with cesium chloride (CsCl) results in photodynamic leaf lesions within 24 h due to an inactivation of uroporphyrinogen III decarboxylase, an enzyme of tetrapyrrole biosynthesis, and transient accumulation of uroporphyrin (ogen). To examine the mechanism of porphyrinogenesis, time kinetics of the accumulating tetrapyrrole intermediates uroporphyrin (ogen) and protochlorophyllide were performed with leaves which were cut from 7-day-old dark-grown barley seedlings and incubated in 15 mM CsCl or water under different light regimes. In the presence of CsCl chlorophyll and carotenoids accumulation was inhibited in the first 24 h of continuous light and the pigment content decreased dramatically during extended illumination. When CsCl=treated leaves were transferred to darkness, accumulated uroporphyrinogen was completely converted to protochlorophyllide. Low temperature fluorescence spectroscopy confirmed that uroporphyrinogen almost completely accumulated in the reduced form. The oxidised form, uroporphyrin, was detectable after 24 h of illumination. The photodynamic leaf lesions became visible at the same time. Protochlorophyllide synthesised from accumulated uroporphyrinogen III in dark incubated leaves had a fluorescence maximum at 635 nm which is indicative for its non-photoconvertible form. Re-illumination of the barley leaves resulted in a rapid degradation of proteins and pigments and an intense lipid peroxidation within less than two hours due to the photodestructive potential of non-metabolised protochlorophyllide.

Mechanisms of regulation and interplastid localization of chlorophyll biosynthesis.

The current concepts of chlorophyll biosynthesis, its interplastid localization, biosynthetic and biochemical heterogeneity, mechanisms of regulation of the key reactions, formation of 5-aminolevulinic acid and incorporation of magnesium into protoporphyrin IX, are reviewed. The literature and author's data demonstrate the existence of in vivo multienzyme systems synthesizing chlorophyll and its precursors as monovinyl and divinyl chemical species. Both types of the multienzyme systems synthesize 5-aminolevulinic acid and regulate this process independently. A hypothesis is considered that the function of the magnesium branch of chlorophyll biosynthesis in vivo is controlled by a mechanism through inhibition of the enzymes by their products because of the limitation of the binding sites for them in the membrane. An additional influence of light on the Mg-chelatase activity not only via the photosynthetic supply with ATP but also through the light-induced synthesis of the enzyme molecules de novo is described. Efficient energy migration from protoporphyrin IX and Mg-protoporphyrin IX (monomethyl ester) molecules to the protochlorophyllide active form detected by the author is discussed considering a close location of these pigments in plastid membranes and the enzymes participating in their formation.

[Localization of Mg-protoporphyrin IX monomethyl ester in chloroplast submembrane particles of barley]. / Lokalizatsiia magnii-protoporfirin IX monometilovogo éfira v submembrannykh chastitsakh khloroplastov iachmenia.

It is shown that Mg-protoporphyrin monomethyl ester as well as immediate chlorophyll precursors are localized in chloroplast submembrane particles, some of them being especially enriched. A conclusion is made that the centers of chlorophyll biosynthesis coupled with chloroplast submembrane particles perform a wide range of biosynthetic reactions beginning at least with Mg-protoporphyrin IX monomethyl ester transformation.

[Effect of chloramphenicol and cycloheximide on synthesis of delta-aminolevulinate dehydratase in green and greening barley shoots]. / Vliianie khloramfenikola i tsiklogeksimida na sintez delta-aminolevulinatdegidratazy v zelenykh i zeleneiushchikh prorostkakh iachemnia.

The synthesis of sigma-aminolevulinate dehydratase (EC 4.2.1.24) in green or greening barley shoots was shown to increase, when the plants were grown on chloramphenicol solutions of varying concentrations for 48 hrs upon illumination. This was evidenced from the increase in the enzyme activity of the chloroplast preparations isolated from the shoots as compared to the controls grown in aqueous media. Similar treatment by cycloheximide resulted in inhibition of the enzyme synthesis as observed in the experiments with green and greening shoots. The activity of porphobilinogenase (the porphobilinogene deaminase and uroporphirinogene III cosynthetase complex) showed similar dependence on the effect of the antibiotics. The results obtained are discussed in terms of localization of the chloroplast enzyme syntheses inside the cell.