Jerzy Kruk

Occurrence, biosynthesis and function of isoprenoid quinones.

Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K(1), a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.

Evidence for Light Wavelength-Specific Photoelectrophysiological Signaling and Memory of Excess Light Episodes in Arabidopsis

This work examines light wavelength–specific electrophysiological signaling and cellular light memory in Arabidopsis. Animals have their network of neurons, synapses, electrophysiological circuits and memory, but plants have their network of chloroplasts (connected by stromules), photoelectrophysiological signals transduced by bundle sheath cells, and cellular light memory. Although light is essential for photosynthesis, excess light can damage the photosynthetic apparatus and deregulate other cellular processes. Thus, protective integrated regulatory responses that can dissipate excess of absorbed light energy and simultaneously optimize photosynthesis and other cellular processes under variable light conditions can prove highly adaptive. Here, we show that the local and systemic responses to an excess light episode are associated with photoelectrophysiological signaling (PEPS) as well as with changes in nonphotochemical quenching and reactive oxygen species levels. During an excess light incident, PEPS is induced by quantum redox changes in photosystem II and in its proximity and/or by changes in glutathione metabolism in chloroplasts. PEPS is transduced, at least in part, by bundle sheath cells and is light wavelength specific. PEPS systemic propagation speed and action potential are dependent on ASCORBATE PEROXIDASE2 function. Excess light episodes are physiologically memorized in leaves, and the cellular light memory effect is specific for an excess of blue (450 nm) and red (650 nm) light of similar energy. It is concluded that plants possess a complex and dynamic light training and memory system that involves quantum redox, reactive oxygen species, hormonal, and PEPS signaling and is used to optimize light acclimation and immune defenses.

Redox Changes of Cytochrome b 559 in the Presence of Plastoquinones

We have found that short chain plastoquinones effectively stimulated photoreduction of the low potential form of cytochrome b 559 and were also active in dark oxidation of this cytochrome under anaerobic conditions in Triton X-100-solubilized photosystem II (PSII) particles. It is also shown that molecular oxygen competes considerably with the prenylquinones in cytochrome b 559 oxidation under aerobic conditions, indicating that both molecular oxygen and plastoquinones could be electron acceptors from cytochromeb 559 in PSII preparations. α-Tocopherol quinone was not active in the stimulation of cytochrome photoreduction but efficiently oxidized it in the dark. Both the observed photoreduction and dark oxidation of the cytochrome were not sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. It was concluded that both quinone-binding sites responsible for the redox changes of cytochrome b 559 are different from either the QA or QB site in PSII and represent new quinone-binding sites in PSII.

Tocochromanols, plastoquinol, and other biological prenyllipids as singlet oxygen quenchers—determination of singlet oxygen quenching rate constants and oxidation products

Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and alpha-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, alpha-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the gamma-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-gamma-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.

Plastoquinol is the Main Prenyllipid Synthesized During Acclimation to High Light Conditions in Arabidopsis and is Converted to Plastochromanol by Tocopherol Cyclase

Plants have evolved various strategies to acclimate to high light conditions at different levels of organization. High light stress stimulates synthesis of different antioxidant enzymes and low molecular weight antioxidants, mainly in chloroplasts. In the present studies we showed that plastoquinol, in addition to alpha-tocopherol, is the main lipid-soluble antioxidant synthesized during acclimation of Arabidopsis plants to high light conditions. The level of plastoquinol increased >10-fold and independently of tocopherols, as revealed using tocopherol biosynthetic mutants. The high light-induced increase in plastoquinol level was mainly attributable to the photochemically non-active fraction of this compound localized in plastoglobuli, which are the storage site of prenyllipids for their antioxidant action. Our data also revealed that tocopherol cyclase is required for plastochromanol biosynthesis from plastoquinol in vivo. Plastochromanol accumulated in increasing amounts in leaves during growth and it was also identified in seeds. The obtained data suggest that plastochromanol may, similarly to other prenyllipids, fulfill antioxidant function in leaves and seeds, especially during aging.

Dark reoxidation of the plastoquinone-pool is mediated by the low-potential form of cytochrome b-559 in spinach thylakoids

We have found that in isolated spinach thylakoids, plastoquinone-pool (PQ-pool), after its photoreduction, undergoes dark-reoxidation with the half-time of τ1/2 = 43 ± 3 s. To explain the observed rates of PQ-pool reoxidation, a nonenzymatic plastoquinol (PQH2) autoxidation under molecular oxygen and an enzymatic oxidation by the low-potential form of cytochrome b-559 (cyt. b-559LP), as the postulated PQ-oxidase in chlororespiration, were investigated. It was found that the autoxidation rate of PQH2 in organic solvents and liposomes was too low to account for the observed oxidation rate of PQH2 in thylakoids. The rate of cyt. b-559LP autoxidation in isolated Photosystem II was found to be similar (τ1/2 = 26 ± 5 s) to that of the PQ-pool. This suggests that the LP form of cyt. b-559 is probably responsible for the PQ-oxidase activity observed during chlororespiration.

Plastoquinol and α-tocopherol quinol are more active than ubiquinol and α-tocopherol in inhibition of lipid peroxidation

Abstract Comparative studies of antioxidant activities of such natural prenyllipids as plastoquinol-9 (PQH2-9), α-tocopherol quinol (α-TQH2), ubiquinol-10 (UQH2-10) and α-tocopherol (α-T) in egg yolk lecithin liposomes have been performed. The investigated compounds showed oxidation under molecular oxygen in the order UQH2-10>α-TQH2>PQH2-9>>α-T. The corresponding second order rate constants have been determined in Tris buffer (pH=6.5) and were 0.413, 0.268, 0.154 and 0.022 M−1/s, respectively. The inhibition order of Fe2+-H2O2 -induced lipid peroxidation, corrected for the amount of prenyllipids oxidized during the initiation period, was α-TQH2>PQH2-9>α-T>UQH2-10 for 5 mol% of the antioxidants content in liposomes. The radicals formed in the initiation phase of the reaction caused oxidation of 27.5–33% α-T, 40–64% UQH2-10, 42–85% PQH2-9 and 43–80% α-TQH2, depending on the antioxidant concentration in liposomes (5–1 mol%, respectively) which reflects approximately their reactivity against radicals derived from the Fenton reaction. The antioxidant activity of the investigated prenylquinols, in relation to the activity of α-T, in natural membranes is discussed.

Riboflavin as a Source of Autofluorescence in Eisenia fetida Coelomocytes

Abstract Immunocompetent cells of earthworms (coelomocytes) contain adherent amoebocytes and large eleocytes (chloragocytes); the latter are filled with numerous granules. We have previously shown that eleocytes of several (but not all) earthworm species exhibit strong autofluorescence detectable by fluorescent microscopy and flow cytometry. In the present article, the molecular origin of eleocytes autofluorescence was elucidated in coelomocytes expelled via dorsal pores in the integument of Eisenia fetida subjected to electric shock (1 min at 4.5 V). Spectrofluorometry (excitation and emission spectra and fluorescence lifetime), together with HPLC analysis of coelomocyte suspensions and supernatants, indicated that riboflavin but not FMN (flavin mononucleotide) or FAD (flavin-adenine dinucleotide) is the main fluorophore responsible for eleocyte fluorescence in this species. Additionally, lipofuscins are suspected to participate in this phenomenon.

Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b559 tobacco mutant.

Light-induced production of superoxide (O2*-) in spinach PSII (photosystem II) membrane particles was studied using EPR spin-trapping spectroscopy. The presence of exogenous PQs (plastoquinones) with a different side-chain length (PQ-n, n isoprenoid units in the side-chain) enhanced O2*- production in the following order: PQ-1>PQ-2>>PQ-9. In PSII membrane particles isolated from the tobacco cyt (cytochrome) b559 mutant which carries a single-point mutation in the beta-subunit and also has a decreased amount of the alpha-subunit, the effect of PQ-1 was less than in the wild-type. The increase in LP (low-potential) cyt b559 content, induced by the incubation of spinach PSII membrane particles at low pH, resulted in a significant increase in O2*- formation in the presence of PQ-1, whereas it had little effect on O2*- production in the absence of PQ-1. The enhancement of O2*- formation induced by PQ-1 was not abolished by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Under anaerobic conditions, dark oxidation of LP cyt b559 increased, as pH was decreased. The presence of molecular oxygen significantly enhanced dark oxidation of LP cyt b559. Based on these findings it is suggested that short-chain PQs stimulate O2*- production via a mechanism that involves electron transfer from Pheo- (pheophytin) to LP cyt b559 and subsequent auto-oxidation of LP cyt b559.

Location of ubiquinone homologues in liposome membranes studied by fluorescence anisotropy of diphenyl-hexatriene and trimethylammonium-diphenyl-hexatriene

The measurements of diphenyl-hexatriene (DPH) and trimethylammonium-diphenyl-hexatriene (TMA-DPH) fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) and egg yolk lecithin (EYL) liposomes containing different concentrations of various ubiquinone (UQ) homologues have been performed. UQ-4 induced the highest DPH anisotropy increase in DPPC liposomes, whereas for higher UQ homologues the anisotropy was lowered with the increase of UQ side-chain length. These differences were less pronounced in EYL liposomes. It was concluded that at a higher content in the membranes (3-4 mol%), the short-chain ubiquinones are arranged parallel to lipid fatty acid chains, whereas long-chain homologues are progressively removed from the lipid acyl chains into the midplane region of the membrane. At the lower (1-2 mol%) concentrations, long-chain quinones seem to be evenly distributed within the membrane, especially in EYL membranes. UQ-10 in EYL liposomes perturbed TMA-DPH to a similar extend as the short-chain ubiquinones indicating that UQ-10 penetrates the interface regions of the membrane where its redox reactions occur. The localization and physical state of UQ-10 in native membranes is discussed.