Lipoxygenase LOX3 Is the Enigmatic Tocopherol Oxidase in Runner Bean (Phaseolus coccineus).

Purification of extracts from the etiolated seedlings of runner bean (Phaseolus coccineus), coupled with mass spectrometry analysis of proteins revealed that the enzyme responsible for tocopherol oxidation activity is lipoxygenase, an enzyme known for enzymatic lipid peroxidation of unsaturated lipids. Biochemical analysis of the activity, along with the expression profile of three LOX isoforms (LOX1, LOX2, LOX3) in various parts of the etiolated seedlings, revealed that LOX3 was the major isoform expressed in the epicotyls, indicating that this isoform was responsible for the tocopherol oxidation activity; in the primary leaves, besides LOX3, the other two isoforms might have also contributed to the activity. The experiments performed in the model systems showed that unsaturated lipids were not required for the tocopherol oxidase activity, but that lipids were necessary to provide an optimal, hydrophobic environment of the substrate for the reaction. The experiments on lipoxygenase and tocopherol oxidase activities in the leaves of light-grown P. coccineus plants during aging and during storage of the extracts from etiolated seedlings showed that the activity of the first reaction decreased considerably faster than the latter, indicating different mechanisms of both reactions performed by the same enzyme. As LOX3 was shown to occur in the apoplast of the related species P. vulgaris, the question as to the physiological function of LOX3 in the tocopherol oxidation activity in P. coccineus is discussed.

The interaction between titanium dioxide nanoparticles and light can have dualistic effects on the physiological responses of plants.

The model plant Arabidopsis thaliana was exposed to combined stress factors, i.e., titanium dioxide nanoparticles (TiNPs) and high light. The concentrations of TiNPs used for irrigation were 250, 500, and 1000 µg/mL. This study shows that TiNPs alter the morphology and nanomechanical properties of chloroplasts in A. thaliana, which leads to a decrease in membrane elasticity. We found that TiNPs contributed to a delay in the thermal response of A. thaliana under dynamic light conditions, as revealed by non-invasive thermal imaging. The thermal time constants of TiNP-treated plants under excessive light are determined, showing a shortening in comparison to control plants. The results indicate that TiNPs may contribute to an alleviation of temperature stress experienced by plants under exposure to high light. In this research, we observed a decline in photosystem II photochemical efficiency accompanied by an increase in energy dissipation upon exposure to TiNPs. Interestingly, concentrations exceeding 250 µg/mL TiNPs appeared to mitigate the effects of high light, as shown by reduced differences in the values of specific OJIP parameters (FV/FM, ABS/RC, DI0/RC, and Pi_Abs) before and after light exposure.

Synthesis of natural polyprenols for the production of biological prenylquinones and tocochromanols.

We elaborate the chemical synthesis of polyprenols by chain lengthening, which is considerably less time-consuming than the other previously described methods. Our method eliminates critical steps requiring low temperature and toxic chemicals, which are difficult to perform in ordinary laboratories. The critical step of acetylene addition in liquid ammonia was replaced by a new approach, namely, the use of sodium acetylide in dimethoxyethane at room temperature, where the reaction is completed within one hour. This method is of general significance as it can also be applied to the synthesis of any other acetylides. Our method provides reasonable yields and can be scaled depending on the requirements. All the reactions were followed by high-performance liquid chromatography, allowing the formation of undesired isomers and other side-products to be controlled. The resulting polyprenols were further used in the synthesis of plastoquinones, although a variety of biological prenylquinones can be synthesized this way. Moreover, we found a new method for the direct formation of tocochromanols (plastochromanols, tocochromanols) from polyprenols and aromatic head groups.

Salinity-induced changes in plastoquinone pool redox state in halophytic Mesembryanthemum crystallinum L.

We have analyzed the effect of salinity on photosystem II (PSII) photochemistry and plastoquinone (PQ) pool in halophytic Mesembryanthemum crystallinum plants. Under prolonged salinity conditions (7 or 10 days of 0.4 M NaCl treatment) we noted an enlarged pool of open PSII reaction centers and increased energy conservation efficiency, as envisaged by parameters of the fast and slow kinetics of chlorophyll a fluorescence. Measurements of oxygen evolution, using 2,6-dichloro-1,4-benzoquinone as an electron acceptor, showed stimulation of the PSII activity due to salinity. In salt-acclimated plants (10 days of NaCl treatment), the improved PSII performance was associated with an increase in the size of the photochemically active PQ pool and the extent of its reduction. This was accompanied by a rise in the NADP+/NADPH ratio. The presented data suggest that a redistribution of PQ molecules between photochemically active and non-active fractions and a change of the redox state of the photochemically active PQ pool indicate and regulate the acclimation of the photosynthetic apparatus to salinity.

Acyl-Nω-methylserotonins and Branched-chain Acylserotonins in Lemon and Other Citrus Seeds-New Lipids with Antioxidant Properties and Potential Pharmacological Applications.

We have found 15 previously unknown compounds in seeds of lemon and other citrus species, such as tangerine, grapefruit and pomelo. The structure of these compounds was characterized by HR-MS spectrometry, fluorescence spectroscopy and chemical synthesis. These compounds were predominantly long-chain (C20-C25), saturated acyl-Nω-methylserotonins with the main contribution of C22 and C24 homologues, usually accounting for about 40% and 30% of all acylserotonins, respectively. The other, previously undescribed, minor compounds were branched-chain acylserotonins, as well as normal-chain acylserotonins, recently found in baobab seed oil. Within the seed, acylserotonins were found nearly exclusively in the inner seed coat, where probably their biosynthesis proceeds. On the other hand, lemon seedlings contained only trace amounts of these compounds that were not found in adult leaves. The compounds identified in the present studies were shown to have antioxidant properties in vitro, using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In the investigated reaction in hexane, Me-C22 and Me-C24-serotonins were less active than n-C22 and n-C24-serotonins and δ-tocopherol, while branched-chain acylserotonins (iso-C21 and -C25) showed higher antioxidant activity than all the normal-chain compounds. On the other hand, all these compounds showed a similar but considerably lower antioxidant activity in acetonitrile than in hexane.

Antioxidant and Neuroprotective Activity of Vitamin E Homologues: In Vitro Study.

Here we present comparative data on the inhibition of lipid peroxidation by a variety of tocochromanols in liposomes. We also show for the first time the potential neuroprotective role of all the vitamin E homologues investigated on the neuronally differentiated human neuroblastoma SH-SY5Y cell line. α-Tocopherol had nearly no effect in the inhibition of lipid peroxidation, while ß-, γ-, and δ-tocopherols inhibited the reaction completely when it was initiated in a lipid phase. Similar effects were observed for tocotrienol homologues. Moreover, in this respect plastochromanol-8 was as effective as ß-, γ-, and δ-tocochromanols. When the prenyllipids were investigated in a 1,1-diphenyl-2-picrylhydrazyl (DPPH) test and incorporated into different lipid carriers, the radical oxidation was most pronounced in liposomes, followed by mixed micelles and the micellar system. When the reaction of tocochromanols was examined in niosomes, the oxidation was most pronounced for α-tocopherol and plastochromanol-8, followed by α-tocotrienol. Next, using retinoic acid-differentiated SH-SY5Y cells, we tested the protective effects of the compounds investigated on hydrogen peroxide (H2O2)-induced cell damage. We showed that tocotrienols were more active than tocopherols in the oxidative stress model. Plastochromanol-8 had a strong inhibitory effect on H2O2-induced lactate dehydrogenase (LDH) release and H2O2-induced decrease in cell viability. The water-soluble α-tocopherol phosphate had neuroprotective effects at all the concentrations analyzed. The results clearly indicate that structural differences between vitamin E homologues reflect their different biological activity and indicate their potential application in pharmacological treatments for neurodegenerative diseases. In this respect, the application of optimal tocochromanol-carrying structures might be critical.

Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny.

Using high-resolution chromatography we resolved monovinyl (MV)- and divinyl (DV)-protochlorophylls (Pchls) and detected all of their side-chain homologues in the inner seed coat of Cucurbita maxima, C. pepo and their varieties. Furthermore, we analyzed other less common representatives of the Cucurbitaceae family that were found to accumulate mostly MV-Pchls. All these species and varieties showed the characteristic composition of individual Pchls. Additionally, we also detected all of the corresponding protopheophytins, which accounted for between 1.1 and 35.5% of Pchls and are supposed to be degradation products of Pchls, formed during seed storage. A pigment composition analysis of C. maxima seedlings performed during deetiolation revealed that chlorophyll (Chl) a content increased gradually, while the levels of Pchl-GG and Chl-GG, a precursor of Chl a, were low and did not change significantly. However, when the seedlings were incubated with the precursor of tetrapyrrole biosynthesis (δ-aminolevulinic acid) before illumination, the Chl-GG content increased dramatically, while synthesis of Chl a was inhibited. These data indicate that in C. maxima seedlings, Chl a is not synthesized from geranylgeranyl-pyrophoshate via Chl-GG, but rather directly from phytyl-pyrophosphate. Phylogenetic analysis of Chl synthase genes revealed that many species, including those of the Cucurbitaceae family, have two or more Chl synthase genes. This suggests that these additional genes, at least in some species, might encode isoforms involved in Pchl synthesis.

Acylserotonins - a new class of plant lipids with antioxidant activity and potential pharmacological applications.

During analysis of components of baobab (Adansonia digitata) seed oil, several new fluorescent compounds were detected in HPLC chromatograms that were not found previously in any seed oils investigated so far. After preparative isolation of these compounds, structural analysis by NMR spectroscopy, UHPLC-HR-MS, GC-FID and spectroscopic methods were applied and allowed identification of these substances as series of N-acylserotonins containing saturated C22 to C26 fatty acids with minor contribution of C27 to C30 homologues. The main component was N-lignocerylserotonin and the content of odd carbon-atom-number fatty acids was unusually high among the homologues. The suggested structure of the investigated compounds was additionally confirmed by their chemical synthesis. Synthetic N-acylserotonins showed pronounced inhibition of membrane lipid peroxidation of liposomes prepared from chloroplast lipids, especially when the peroxidation was initiated by a water-soluble azo-initiator, AIPH. Comparative studies of the reaction rate constants of the N-acylserotonins and tocopherols with a stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in solvents of different polarity revealed that N-acylserotonins showed similar activity to δ-tocopherol in this respect. The described compounds have been not reported before either in plants or in animals. This indicates that we have identified a new class of plant lipids with antioxidant properties that could have promising pharmacological activities.

Photocatalytic LPOR forms helical lattices that shape membranes for chlorophyll synthesis.

Chlorophyll biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme light-dependent protochlorophyllide oxidoreductase (LPOR) captures photons to catalyse the penultimate reaction: the reduction of a double bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete three-dimensional structure of LPOR, the higher-order architecture of LPOR oligomers and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the co-factor NADPH, remain unknown. Here, we report the atomic structure of LPOR assemblies by electron cryo-microscopy. LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolamellar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Moreover, our structure of the catalytic site challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants, orchestrated by LPOR.

Singlet oxygen oxidation products of carotenoids, fatty acids and phenolic prenyllipids.

Singlet oxygen (1O2) is the major reactive oxygen species ROS causing photooxidative stress in plants which is formed predominantly in the reaction center of photosystem II during photosynthesis. To avoid deleterious effects of 1O2 oxygen on photosynthetic membrane components, plant synthesize a variety of 1O2 quenchers of lipophilic character, such as carotenoids or phenolic prenyllipids (tocopherols, plastochromanol-8, plastoquinol). In the process of chemical quenching of 1O2 by the antioxidants, both short-lived products, such as oxidized carotenoids, or relative long-lived compounds, such as oxidized phenolic prenyllipids are formed. The other target of 1O2 are unsaturated fatty acids of membrane lipids that undergo peroxidation as a result of the reaction. Some of the 1O2 oxidation products, like ß-cyclocitral can be components of 1O2-signallingsignaling pathway leading to acclimatory responses of plants, while some others further fulfill antioxidant functions, like hydroxy-plastochromanol or hydroxy-plastoquinol. As most of the 1O2 oxidation products are specific compounds formed only as a results of 1O2 action, they can be very useful, specific molecular markers of 1O2-dependent oxidative stress in vivo.

The inhibitor-evoked shortage of tocopherol and plastoquinol is compensated by other antioxidant mechanisms in Chlamydomonas reinhardtii exposed to toxic concentrations of cadmium and chromium ions.

One of the major mechanisms of heavy metal toxicity is the induction of oxidative stress. Redox-active heavy metals, like chromium, can induce it directly, whereas redox-inactive metals, like cadmium, play an indirect role in the generation of reactive oxygen species (ROS). Living organisms defend themselves against oxidative stress taking advantage of low-molecular-weight antioxidants and ROS-detoxifying enzymes. Tocopherols and plastoquinol are important plastid prenyllipid antioxidants, playing a role during acclimation of Chlamydomonas reinhardtii to heavy metal-induced stress. However, partial inhibition of synthesis of these prenyllipids by pyrazolate did not decrease the tolerance of C. reinhardtii to Cr- and Cd-induced stress, suggesting redundancy between antioxidant mechanisms. To verify this hypothesis we have performed comparative analyses of growth, photosynthetic pigments, low-molecular-weight antioxidants (tocopherols, plastoquinol, plastochromanol, ascorbate, soluble thiols, proline), activities of the ascorbate peroxidase (APX), catalase and superoxide dismutase (SOD) and cumulative superoxide production in C. reinhardtii exposed to Cd2+ and Cr2O72- ions in the presence or absence of pyrazolate. The decreased α-tocopherol and plastoquinol content resulted in the increase in superoxide generation and APX activity in pyrazolate-treated algae. The application of heavy metal ions and pyrazolate had a pronounced impact on Asc and total thiol content, as well as SOD and APX activities (the latter only in Cd-exposed cultures), when compared with algae grown in the presence of heavy metal ions or pyrazolate alone. The superoxide production in cultures exposed to heavy metal ions and pyrazolate decreased when compared to the cultures exposed to either heavy metal ions or an inhibitor alone.

Identification of new fluorophores in coelomic fluid of Eisenia andrei earthworms.

Coelomic fluid of Eisenia andrei contains a number of UV-fluorescent compounds. In the present study we have found that four of these compounds showed identical fluorescence excitation and emission maxima at 310 nm and 364 nm, respectively, suggesting they share the same chromophore. NMR and HR-MS spectroscopy of the most abundant fluorophore reavealed that its molecule is composed of two quinazoline-2,4-dione rings connected by spermine linker. This compound was earlier indentified in Eisenia andrei as SP-8203. Moreover, we have identified the structure of the two other fluorophores, one differing from SP-8203 by the absence of N-acetyl group, the compound not reported in any other organisms before, and the other already found in E. fetida and regarded as species specific. However, our results indicate that this metabolite is also present in E. andrei in significant amounts. The possible origin and function of these new metabolites is discussed.

Improving photosynthesis, plant productivity and abiotic stress tolerance - current trends and future perspectives.

With unfavourable climate changes and an increasing global population, there is a great need for more productive and stress-tolerant crops. As traditional methods of crop improvement have probably reached their limits, a further increase in the productivity of crops is expected to be possible using genetic engineering. The number of potential genes and metabolic pathways, which when genetically modified could result in improved photosynthesis and biomass production, is multiple. Photosynthesis, as the only source of carbon required for the growth and development of plants, attracts much attention is this respect, especially the question concerning how to improve CO2 fixation and limit photorespiration. The most promising direction for increasing CO2 assimilation is implementating carbon concentrating mechanisms found in cyanobacteria and algae into crop plants, while hitherto performed experiments on improving the CO2 fixation versus oxygenation reaction catalyzed by Rubisco are less encouraging. On the other hand, introducing the C4 pathway into C3 plants is a very difficult challenge. Among other points of interest for increased biomass production is engineering of metabolic regulation, certain proteins, nucleic acids or phytohormones. In this respect, enhanced sucrose synthesis, assimilate translocation to sink organs and starch synthesis is crucial, as is genetic engineering of the phytohormone metabolism. As abiotic stress tolerance is one of the key factors determining crop productivity, extensive studies are being undertaken to develop transgenic plants characterized by elevated stress resistance. This can be accomplished due to elevated synthesis of antioxidants, osmoprotectants and protective proteins. Among other promising targets for the genetic engineering of plants with elevated stress resistance are transcription factors that play a key role in abiotic stress responses of plants. In this review, most of the approaches to improving the productivity of plants that are potentially promising and have already been undertaken are described. In addition to this, the limitations faced, potential challenges and possibilities regarding future research are discussed.

Phytohormones as targets for improving plant productivity and stress tolerance.

In this review, we summarize the results of experiments that lead to altered levels of phytohormones in transgenic plants to improve plant productivity. The available data indicate that manipulating the level of phytohormones might also be a promising way to enhance the environmental stress tolerance of crop plants. In the regulation of the level of phytohormones, both biosynthesis and their catabolism pathways can be targeted for engineering purposes. Moreover, the signaling pathways of phytohormones should explored in this respect. In genetic modifications, conditional promoters must be developed to avoid undesired effects on growth. In order to find a practical application, the effects of genetic modifications should be further verified under field conditions and over a longer time scale.

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis.

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (1 O2 ) formed during high light stress in higher plants. Although quenching of 1 O2 by prenylquinols has been previously studied, direct evidence for chemical quenching of 1 O2 by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol-9 (PQH2 -9) in chemical quenching of 1 O2 was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH2 -9 and plastochromanol-8 biosynthesis. In this work, direct evidence for chemical quenching of 1 O2 by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of 1 O2 was associated with consumption of PQH2 -9 and formation of its various oxidized forms. Oxidation of PQH2 -9 by 1 O2 leads to plastoquinone-9 (PQ-9), which is subsequently oxidized to hydroxyplastoquinone-9 [PQ(OH)-9]. We provide here evidence that oxidation of PQ(OH)-9 by 1 O2 results in the formation of trihydroxyplastoquinone-9 [PQ(OH)3 -9]. It is concluded here that PQH2 -9 serves as an efficient 1 O2 chemical quencher in Arabidopsis, and PQ(OH)3 -9 can be considered as a natural product of 1 O2 reaction with PQ(OH)-9. The understanding of the mechanisms underlying 1 O2 chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.

Lack of tocopherols influences the PSII antenna and the functioning of photosystems under low light.

As tocopherols are expected to protect PSII against toxic singlet oxygen it is surprising that the null tocopherol mutant vte1 has been reported to show only a weak enhancement of photosystem II photoinhibition under high irradiance. Based on the view that singlet oxygen is formed also in unstressed conditions, such as low light (LL), we hypothesized that some defense strategies are activated in vte1 in these light conditions. In support for that we noted several symptoms of stress at PSII in the mutant under LL, by means of parameters of fast and slow kinetics of chlorophyll fluorescence and of changes in the relative contribution of PSII antenna in comparison to those of PSI. This was associated with a lower extent of phosphorylation of PSII core proteins (D1 and CP43). PSII RCs do not totally recover from stress in vte1 even after the nocturnal phase. As a clear compensation for the impeded performance of PSII in the vte1 we noted an increased quantum efficiency of PSI. A pronounced changes between WT and the vte1 mutant were also related to conformation of LHCII at the beginning of photoperiod, suggesting the absence of LHCII trimers in the mutant. The thylakoids thickness was similar in WT and vte1 under LL, but a pronounced unstacking of thylakoids was evoked by HL only in vte1. In conclusion, we postulate that action of 1O2 on PSII in vte1 leads to some permanent damage at PSII core and at LHCII already under LL.

[Genetic engineering as a method for the improvement of photosynthesis]. / Perspektywy wykorzystania inzynierii genetycznej do zwiekszenia wydajnosci fotosyntezy

The significant increase in crop productivity occurred in the second half o the 20th century. However, it is thought that nowadays yield of main crop species reached its maximum. As we expect that the demand for plant products is going to increase during next century, it is necessary to develop new methods for yield improvement, other than traditional breeding. The redesign of photosynthesis using genetic engineering is one of the approaches postulated. The present article covers the main directions of research aimed to increase photosynthetic efficiency. The research covered by this review are: improvement of light capture, improvement of Rubisco and the regeneration phase of Calvin cycle, introducing carbon concentrating mechanisms to main crop species and reducing loss caused by photorespiration.

Novel and rare prenyllipids - Occurrence and biological activity.

The data presented indicate that there is a variety of unique prenyllipids, often of very limited taxonomic distribution, whose origin, biosynthesis, metabolism and biological function deserves to be elucidated. These compounds include tocoenols, tocochromanol esters, tocochromanol acids, plastoquinones and ubiquinones. Additionally, based on the available data, it can be assumed that there are still unrecognized prenyllipids, like prenylquinols fatty acid esters of the hydroquinone ring, including prenylquinol phosphates, and others, whose biological function might be of great importance. Our knowledge of these compounds is not only important from the scientific point of view, but may also be of practical significance to medicine, pharmacy or cosmetics.

RubisCO Early Oxygenase Activity: A Kinetic and Evolutionary Perspective.

RubisCO (D-ribulose 1,5-bisphosphate carboxylase/oxygenase) is Earth's main enzyme responsible for CO2 fixation via carboxylation of ribulose-1,5-bisphosphate (RuBP) into organic matter. Besides the carboxylation reaction, RubisCO also catalyzes the oxygenation of RuBP by O2 , which is probably as old as its carboxylation properties. Based on molecular phylogeny, the occurrence of the reactive oxygen species (ROS)-removing system and kinetic properties of different RubisCO forms, we postulated that RubisCO oxygenase activity appeared in local microoxic areas, yet before the appearance of oxygenic photosynthesis. Here, in reviewing the literature, we present a novel hypothesis: the RubisCO early oxygenase activity hypothesis. This hypothesis may be compared with the exaptation hypothesis, according to which latent RubisCO oxygenase properties emerged later during the oxygenation of the Earth's atmosphere. The reconstruction of ancestral RubisCO forms using ancestral sequence reconstruction (ASR) techniques, as a promising way for testing of RubisCO early oxygenase activity hypothesis, is presented.