Unravelling the Mechanisms of Heavy Metal Tolerance: Enhancement in Hydrophilic Antioxidants and Major Antioxidant Enzymes Is Not Crucial for Long-Term Adaptation to Copper in Chlamydomonas reinhardtii.

Understanding of the mechanisms of heavy metal tolerance in algae is important for obtaining strains that can be applied in wastewater treatment. Cu is a redox-active metal directly inducing oxidative stress in exposed cells. The Cu-tolerant Chlamydomonas reinhardtii strain Cu2, obtained via long-term adaptation, displayed increased guaiacol peroxidase activity and contained more lipophilic antioxidants, i.e., α-tocopherol and plastoquinol, than did non-tolerant strain N1. In the present article, we measured oxidative stress markers; the content of ascorbate, soluble thiols, and proline; and the activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in N1 and Cu2 strains grown in the absence or presence of excessive Cu. The Cu2 strain displayed less pronounced lipid peroxidation and increased APX activity compared to N1. The amount of antioxidants was similar in both strains, while SOD and CAT activity was lower in the Cu2 strain. Exposure to excessive Cu led to a similar increase in proline content in both strains and a decrease in ascorbate and thiols, which was more pronounced in the N1 strain. The Cu2 strain was less tolerant to another redox-active heavy metal, namely chromium. Apparently other mechanisms, probably connected to Cu transport, partitioning, and chelation, are more important for Cu tolerance in Cu2 strain.

The role of antioxidant response and nonphotochemical quenching of chlorophyll fluorescence in long-term adaptation to Cu-induced stress in Chlamydomonas reinhardtii.

Copper is an essential micronutrient, but at supraoptimal concentrations it is also highly toxic, inducing oxidative stress and disrupting photosynthesis. The aim of the present study was to analyze selected protective mechanisms in strains of Chlamydomonas reinhardtii adapted and not adapted for growth in the presence of elevated copper concentrations. Two algal lines (tolerant and non-tolerant to high Cu2+ concentrations) were used in experiments to study photosynthetic pigment content, peroxidase activity, and non-photochemical quenching. The content of prenyllipids was studied in four different algal lines (two of the same as above and two new ones). The copper-adapted strains contained about 2.6 times more α-tocopherol and plastoquinol and about 1.7 times more total plastoquinone than non-tolerant strains. Exposure to excess copper led to oxidation of the plastoquinone pool in non-tolerant strains, whereas this effect was less pronounced or did not occur in copper-tolerant strains. Peroxidase activity was approximately 1.75 times higher in the tolerant strain than in the non-tolerant one. The increase in peroxidase activity in the tolerant strain was less pronounced when the algae were grown in dim light. In the tolerant line nonphotochemical quenching was induced faster and was usually about 20-30% more efficient than in the non-tolerant line. The improvement of antioxidant defense and photoprotection may be important factors in the evolutionary processes leading to tolerance to heavy metals.

Impact of cytotoxic plant naphthoquinones, juglone, plumbagin, lawsone and 2-methoxy-1,4-naphthoquinone, on Chlamydomonas reinhardtii reveals the biochemical mechanism of juglone toxicity by rapid depletion of plastoquinol.

Hydrophilic, untethered 1,4-naphthoquinones (1,4-NQs) are plant secondary metabolites that are often excreted into the environment and play a role in various plant-microbial, plant-fungal, plant-insect and plant-plant interactions. The biological activity of 1,4-NQs is mainly related to their redox properties, i.e. the ability to undergo redox cycling in cells. These compounds may also undergo electrophilic addition to thiol-containing compounds. The aim of this study was to compare the impact of juglone, plumbagin, lawsone and 2-methoxy-1,4-naphthoquinone (2-met-NQ) on the antioxidant response of the green microalga Chlamydomonas reinhardtii. The algae were incubated with the examined compounds under low light for 6 h and the content of photosynthetic pigments, prenyllipid antioxidants, ascorbate, soluble thiols, proline, and superoxide dismutase activity was assessed. To examine the interaction between photosynthetic activity and naphthoquinone toxicity, we carried out the second experiment, in which C. reinhardtii was incubated with 1,4-NQs for 1 h under high light or in darkness. The pro-oxidant action of the examined 1,4-NQs depended on their reduction potentials, which decrease in order: juglone > plumbagin > 2-met-NQ > lawsone. Lawsone did not display pro-oxidant properties. Exposure to high light strongly enhanced the pro-oxidant effect of juglone, plumbagin, and 2-met-NQ, which is thought to result from the interception of the electrons from photosynthetic electron transfer chain. Only juglone was able to cause a fast depletion of plastoquinol, which may be an important mode of action of this allelochemical, responsible for its high toxicity to plants.

Modifications of Phytohormone Metabolism Aimed at Stimulation of Plant Growth, Improving Their Productivity and Tolerance to Abiotic and Biotic Stress Factors.

Due to the growing human population, the increase in crop yield is an important challenge for modern agriculture. As abiotic and biotic stresses cause severe losses in agriculture, it is also crucial to obtain varieties that are more tolerant to these factors. In the past, traditional breeding methods were used to obtain new varieties displaying demanded traits. Nowadays, genetic engineering is another available tool. An important direction of the research on genetically modified plants concerns the modification of phytohormone metabolism. This review summarizes the state-of-the-art research concerning the modulation of phytohormone content aimed at the stimulation of plant growth and the improvement of stress tolerance. It aims to provide a useful basis for developing new strategies for crop yield improvement by genetic engineering of phytohormone metabolism.

Heavy metal-induced stress in eukaryotic algae-mechanisms of heavy metal toxicity and tolerance with particular emphasis on oxidative stress in exposed cells and the role of antioxidant response.

Heavy metals is a collective term describing metals and metalloids with a density higher than 5 g/cm3. Some of them are essential micronutrients; others do not play a positive role in living organisms. Increased anthropogenic emissions of heavy metal ions pose a serious threat to water and land ecosystems. The mechanism of heavy metal toxicity predominantly depends on (1) their high affinity to thiol groups, (2) spatial similarity to biochemical functional groups, (3) competition with essential metal cations, (4) and induction of oxidative stress. The antioxidant response is therefore crucial for providing tolerance to heavy metal-induced stress. This review aims to summarize the knowledge of heavy metal toxicity, oxidative stress and antioxidant response in eukaryotic algae. Types of ROS, their formation sites in photosynthetic cells, and the damage they cause to the cellular components are described at the beginning. Furthermore, heavy metals are characterized in more detail, including their chemical properties, roles they play in living cells, sources of contamination, biochemical mechanisms of toxicity, and stress symptoms. The following subchapters contain the description of low-molecular-weight antioxidants and ROS-detoxifying enzymes, their properties, cellular localization, and the occurrence in algae belonging to different clades, as well as the summary of the results of the experiments concerning antioxidant response in heavy metal-treated eukaryotic algae. Other mechanisms providing tolerance to metal ions are briefly outlined at the end.

Antioxidant and Signaling Role of Plastid-Derived Isoprenoid Quinones and Chromanols.

Plant prenyllipids, especially isoprenoid chromanols and quinols, are very efficient low-molecular-weight lipophilic antioxidants, protecting membranes and storage lipids from reactive oxygen species (ROS). ROS are byproducts of aerobic metabolism that can damage cell components, they are also known to play a role in signaling. Plants are particularly prone to oxidative damage because oxygenic photosynthesis results in O2 formation in their green tissues. In addition, the photosynthetic electron transfer chain is an important source of ROS. Therefore, chloroplasts are the main site of ROS generation in plant cells during the light reactions of photosynthesis, and plastidic antioxidants are crucial to prevent oxidative stress, which occurs when plants are exposed to various types of stress factors, both biotic and abiotic. The increase in antioxidant content during stress acclimation is a common phenomenon. In the present review, we describe the mechanisms of ROS (singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radical) production in chloroplasts in general and during exposure to abiotic stress factors, such as high light, low temperature, drought and salinity. We highlight the dual role of their presence: negative (i.e., lipid peroxidation, pigment and protein oxidation) and positive (i.e., contribution in redox-based physiological processes). Then we provide a summary of current knowledge concerning plastidic prenyllipid antioxidants belonging to isoprenoid chromanols and quinols, as well as their structure, occurrence, biosynthesis and function both in ROS detoxification and signaling.

The xanthophyll cycle in diatom Phaeodactylum tricornutum in response to light stress.

Chosen aspects of the functioning of diadinoxanthin cycle in a model diatom Phaeodactylum tricornutum grown under low light conditions (LL) and under high light conditions (HL), which cause activation of violaxanthin cycle, were examined. Heterogeneity of the kinetics of diadinoxanthin ↔ diatoxanthin conversions regulated by de-epoxidase/epoxidase enzymes was detected. Three different rates of diadinoxanthin de-epoxidation (τ > 20 min, 5 min > τ > 1.5 min and τ ≤ 1 min) were observed. Appearance and contribution of these phases depended on the light conditions and xanthophylls subpopulations in membranes. Moreover, diadinoxanthin de-epoxidation was postulated to occur in darkness and its rate was estimated to be almost two times faster (τ ≈ 14 min) than diatoxanthin-epoxidation in LL- and HL-grown diatoms collected after the dark phase of the photoperiod and exposed to very high light and subsequent darkness. The level of lipid hydroperoxides and the expression of genes encoding xanthophyll cycle enzymes was measured. Our observations suggest that isoforms of these enzymes may participate in carotenoid synthesis or be exclusively involved in xanthophyll conversions. Violaxanthin cycle pigments present in HL-acclimated diatoms change thermodynamic properties of thylakoid membranes. Zeaxanthin is known to localize preferentially in the inner part of the lipid bilayer and diatoxanthin in its outer part. The different localization of these pigments probably decide about their complementary action in protection of the membranes against reactive oxygen species.

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.

Practical aspects of the measurements of non-photochemical chlorophyll fluorescence quenching in green microalgae Chlamydomonas reinhardtii using Open FluorCam.

Methods of chlorophyll fluorescence measurements are widely used in the research on photosynthesis and ecophysiology of plants and algae. Among them, a very popular technique is pulse-amplitude-modulation (PAM) flourometry, which is simple to carry out, fast and non-invasive. However, this method is also prone to generate artifacts if the experiments were not planned and executed properly. Application of this technique to algae brings additional complications, which need to be taken into consideration. Some of them are connected with sample preparation and setting of the protocols used, while another origin from the differences in the photosynthetic apparatus and regulation of photosynthesis in various algal groups when compared to vascular plants. In the present paper, some important practical aspects concerning PAM fluorometry measurements in the green microalga Chlamydomonas reinhardtii have been described, including the equipment settings and sample preparation. The impact of growth conditions, such as light, temperature and medium type on the induction of non-photochemical quenching of chlorophyll fluorescence have been also tackled, as well as the question of state transitions occurring in darkness.

Target genes for plant productivity improvement.

The use of chemical fertilizers and pesticides, as well as the development of high-yielding varieties enabled substantial increase in crop productivity during the 20th century. However, the increase in yield over the last two decades has been slower. It is thought that further improvement in productivity of the major crop species using traditional cultivation methods is limited. Therefore, the use of genetic engineering seems to be a promising approach. There is ongoing research concerning genes that have an impact on plant growth, development and yield. The proteins and miRNAs encoded by these genes participate in a variety of processes, such as growth regulation, assimilate transport and partitioning as well as macronutrient uptake and metabolism. This paper presents the major directions in research concerning genes that may be targets of genetic engineering aimed to improve plant productivity.

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.

[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.

The oxidative stress in allelopathy: Participation of prenyllipid antioxidants in the response to juglone in Chlamydomonas reinhardtii.

Allelopathy is a phenomenon, where one species releases compounds able to inhibit the growth of other species. Juglone, 5-hydroxy-1,4-naphtoquinone, is an allelochemical produced by walnut trees. The main mode of juglone toxicity is the formation of semiquinone radicals, able to reduce O2 to superoxide. Prenyllipid antioxidants such as tocopherol and plastoquinone are important for antioxidant defense in photosynthetic organisms. Here we assess their participation in the response to juglone. The impact of 20 µM juglone on the content of photosynthetic pigments and prenyllipid antioxidants in green microalga Chlamydomonas reinhardtii was measured over an incubation period of 7.5 h in low light and over 40 min under high light or in darkness. The decrease in pigment and prenyllipid content, accompanied by an increase in lipid hydroperoxides was observed over a longer incubation period with juglone. Simultaneous exposure to high light and juglone led to a pronounced decrease in carotenoids and prenyllipids, while there was no decrease in high light alone and no decrease or only a slight decrease in the series with juglone alone. The fact that semiquinone radicals are generated in juglone-exposed cells was confirmed using EPR spectroscopy. This article also shows that C. reinhardtii may be a suitable model for studies on some modes of phytotoxic action of allelochemicals.

Vitamin E - Occurrence, Biosynthesis by Plants and Functions in Human Nutrition.

OBJECTIVE: This review examines various aspects of vitamin E, both in plant metabolism and with regard to its importance for human health. Vitamin E is the collective name of a group of lipidsoluble compounds, chromanols, which are widely distributed in the plant kingdom. Their biosynthetic pathway, intracellular distribution and antioxidant function in plants are well recognized, although their other functions are also considered. CONCLUSION: Analytical methods for the determination of vitamin E are discussed in detail. Furthermore, the vitamin E metabolism and its antioxidant action in humans are described. Other nonantioxidant functions of vitamin E are also presented, such as its anti-inflammatory effects, role in the prevention of cardiovascular diseases and cancer, as well as its protective functions against neurodegenerative and other diseases.

Tocopherol Cyclases-Substrate Specificity and Phylogenetic Relations.

In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms.

Powered by light: Phototrophy and photosynthesis in prokaryotes and its evolution.

Photosynthesis is a complex metabolic process enabling photosynthetic organisms to use solar energy for the reduction of carbon dioxide into biomass. This ancient pathway has revolutionized life on Earth. The most important event was the development of oxygenic photosynthesis. It had a tremendous impact on the Earth's geochemistry and the evolution of living beings, as the rise of atmospheric molecular oxygen enabled the development of a highly efficient aerobic metabolism, which later led to the evolution of complex multicellular organisms. The mechanism of photosynthesis has been the subject of intensive research and a great body of data has been accumulated. However, the evolution of this process is not fully understood, and the development of photosynthesis in prokaryota in particular remains an unresolved question. This review is devoted to the occurrence and main features of phototrophy and photosynthesis in prokaryotes. Hypotheses concerning the origin and spread of photosynthetic traits in bacteria are also discussed.

Physiological characterization of Chlamydomonas reinhardtii acclimated to chronic stress induced by Ag, Cd, Cr, Cu and Hg ions.

Acclimation to heavy metal-induced stress is a complex phenomenon. Among the mechanisms of heavy metal toxicity, an important one is the ability to induce oxidative stress, so that the antioxidant response is crucial for providing tolerance to heavy metal ions. The effect of chronic stress induced by ions of five heavy metals, Ag, Cu, Cr (redox-active metals) Cd, Hg (nonredox-active metals) on the green microalga Chlamydomonas reinhardtii was examined at two levels - the biochemical (content of photosynthetic pigments and prenyllipid antioxidants, lipid peroxidation) and the physiological (growth rate, photosynthesis and respiration rates, induction of nonphotochemical quenching of chlorophyll fluorescence). The expression of the genes which encode the enzymes participating in the detoxification of reactive oxygen species (APX1, CAT1, FSD1, MSD1) was measured. The other gene measured was one required for plastoquinone and α-tocopherol biosynthesis (VTE3). The application of heavy metal ions partly inhibited growth and biosynthesis of chlorophyll. The growth inhibition was accompanied by enhanced lipid peroxidation. An increase in the content of prenyllipid antioxidants was observed in cultures exposed to Cr2O7(2-), Cd(2+) (α- and γ-tocopherol and plastoquinone) and Cu(2+) (only tocopherols). The induction of nonphotochemical quenching was enhanced in cultures exposed to Cu(2+), Cr2O7(2-) and Cd(2+), as compared to the control. Chronic heavy metal-induced stress led to changes in gene expression dependent on the type and concentration of heavy metal ions. The up-regulation of antioxidant enzymes was usually accompanied by the up-regulation of the VTE3 gene.

Function of isoprenoid quinones and chromanols during oxidative stress in plants.

Isoprenoid quinones and chromanols in plants fulfill both signaling and antioxidant functions under oxidative stress. The redox state of the plastoquinol pool (PQ-pool), which is modulated by interaction with reactive oxygen species (ROS) during oxidative stress, has a major regulatory function in both short- and long-term acclimatory responses. By contrast, the scavenging of ROS by prenyllipids affects signaling pathways where ROS play a role as signaling molecules. As the primary antioxidants, isoprenoid quinones and chromanols are synthesized under high-light stress in response to any increased production of ROS. During photo-oxidative stress, these prenyllipids are continuously synthesized and oxidized to other compounds. In turn, their oxidation products (hydroxy-plastochromanol, plastoquinol-C, plastoquinone-B) can still have an antioxidant function. The oxidation products of isoprenoid quinones and chromanols formed specifically in the face of singlet oxygen, can be indicators of singlet oxygen stress.

New prenyllipid metabolites identified in Arabidopsis during photo-oxidative stress.

In the present study, we have identified new prenyllipid metabolites formed during high light stress in Arabidopsis thaliana, whose origin and function remained unknown so far. It was found that plastoquinone-C accumulates mainly in the reduced form under high light conditions, as well as during short-term excess light illumination both in the wild-type and tocopherol biosynthetic vte1 mutant, suggesting that plastoquinone-C, a singlet oxygen-derived prenyllipid, is reduced in chloroplasts by photosystem II or enzymatically, outside thylakoids. Plastoquinone-B, a fatty acid ester of plastoquinone-C, was identified for the first time in Arabidopsis in high light grown wild-type plants and during short-time, excess light illumination of the wild-type plants and the vte1 mutant. The gene expression analysis showed that vte2 gene is most pronouncedly up-regulated among the prenyllipid biosynthetic genes under high light and induction of its expression is mainly caused by an increased level of singlet oxygen, as was demonstrated in experiments with D2 O-treated plants under excess light conditions.

Physiological and antioxidant responses of two accessions of Arabidopsis thaliana in different light and temperature conditions.

During their lifetime, plants need to adapt to a changing environment, including light and temperature. To understand how these factors influence plant growth, we investigated the physiological and antioxidant responses of two Arabidopsis accessions, Shahdara (Sha) from the Shahdara valley (Tajikistan, Central Asia) in a mountainous area and Lovvik-5 (Lov-5) from northern Sweden to different light and temperature conditions. These accessions originate from different latitudes and have different life strategies, both of which are known to be influenced by light and temperature. We showed that both accessions grew better in high-light and at a lower temperature (16°C) than in low light and at 23°C. Interestingly, Sha had a lower chlorophyll content but more efficient non-photochemical quenching than Lov-5. Sha, also showed a higher expression of vitamin E biosynthetic genes. We did not observe any difference in the antioxidant prenyllipid level under these conditions. Our results suggest that the mechanisms that keep the plastoquinone (PQ)-pool in more oxidized state could play a role in the adaptation of these accessions to their local climatic conditions.