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.

Effectiveness of behaviour change techniques on lifestyle interventions of patients with a high risk of developing cardiovascular disease. Using a qualitative approach.

This study aimed to identify key active ingredients on the maintenance of behaviour change for lifestyle interventions of patients with a high risk of developing cardiovascular disease (CVD) who participated in a MOtiVational intErviewing InTervention (MOVE IT) randomised control trial (RCT). A process evaluation was carried out using focus groups. Twenty-six participants of the MOVE IT RCT were purposively recruited and split into six focus groups. Four groups had attended six or more sessions of the intensive phase (completers) and two groups had withdrawn before the end of the intensive phase or had not attended any sessions (non-completers). Focus groups were audio recorded, transcribed verbatim and analysed inductively using thematic analysis. Three overall themes were generated from the six focus groups: (a) long-term benefits from diet and physical activity education, (b) group versus individual structure and adherence and (c) impact on health beliefs and risk of CVD. A fourth theme was generated from the two groups of non-completers only: (d) need for professional rapport building and feedback. We found that the key active ingredients for effective behavioural change in lifestyle interventions are having well-developed rapport between facilitators and patients; and providing alternative forms of feedback to encourage maintenance of behaviour change. Furthermore, such programmes also need to have established and strong relationships with associated health professionals (i.e. the General Practitioner) to increase participation and maintenance of engagement.

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.

Isolation and purification of all-trans diadinoxanthin and all-trans diatoxanthin from diatom Phaeodactylum tricornutum.

Two diatom-specific carotenoids are engaged in the diadinoxanthin cycle, an important mechanism which protects these organisms against photoinhibition caused by absorption of excessive light energy. A high-performance and economical procedure of isolation and purification of diadinoxanthin and diatoxanthin from the marine diatom Phaeodactylum tricornutum using a four-step procedure has been developed. It is based on the use of commonly available materials and does not require advanced technology. Extraction of pigments, saponification, separation by partition and then open column chromatography, which comprise the complete experimental procedure, can be performed within 2 days. This method allows HPLC grade diadinoxanthin and diatoxanthin of a purity of 99 % or more to be obtained, and the efficiency was estimated to be 63 % for diadinoxanthin and 73 % for diatoxanthin. Carefully selected diatom culture conditions as well as analytical ones ensure highly reproducible performance. A protocol can be used to isolate and purify the diadinoxanthin cycle pigments both on analytical and preparative scale.

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.

Photosynthetic Pigments in Diatoms.

Photosynthetic pigments are bioactive compounds of great importance for the food, cosmetic, and pharmaceutical industries. They are not only responsible for capturing solar energy to carry out photosynthesis, but also play a role in photoprotective processes and display antioxidant activity, all of which contribute to effective biomass and oxygen production. Diatoms are organisms of a distinct pigment composition, substantially different from that present in plants. Apart from light-harvesting pigments such as chlorophyll a, chlorophyll c, and fucoxanthin, there is a group of photoprotective carotenoids which includes ß-carotene and the xanthophylls, diatoxanthin, diadinoxanthin, violaxanthin, antheraxanthin, and zeaxanthin, which are engaged in the xanthophyll cycle. Additionally, some intermediate products of biosynthetic pathways have been identified in diatoms as well as unusual pigments, e.g., marennine. Marine algae have become widely recognized as a source of unique bioactive compounds for potential industrial, pharmaceutical, and medical applications. In this review, we summarize current knowledge on diatom photosynthetic pigments complemented by some new insights regarding their physico-chemical properties, biological role, and biosynthetic pathways, as well as the regulation of pigment level in the cell, methods of purification, and significance in industries.

Expression of three diadinoxanthin de-epoxidase genes of Phaeodacylum tricornutum in Escherichia coli Origami b and BL21 strain.

In the diadinoxanthin cycle the epoxy group is removed from diadinoxanthin and diatoxanthin is created. This conversion takes place e.g. in diatoms with the involvement of the enzyme diadinoxanthin de-epoxidase. In one of the diatom species, Phaeodactylum tricornutum (CCAP 1055/1 strain with genome sequenced) three de-epoxidase genes (PtVDE, PtVDL1, PtVDL2) have been identified, but only one of them (PtVDE) corresponds to violaxanthin de-epoxidase, an enzyme which is commonly found in higher plants. In these studies, the expression of two de-epoxidase genes of another Phaeodactylum tricornutum strain (UTEX 646), which is commonly used in diatom studies, were obtained in Origami b and BL21 E. coli strains. The molecular masses of the mature proteins are about 49 kDa and 60 kDa, respectively, for VDE and VDL2. Both enzymes are active with violaxanthin as a substrate.

Temperature effect on growth, and selected parameters of Phaeodactylum tricornutum in batch cultures.

The effect of optimal and stress temperatures on the growth kinetics of the Phaeodactylum tricornutum CCAP/1055/1 strain (a model diatom with a known genome sequence) in batch cultures was examined. The analysis of the obtained results showed two phases of culture growth. There were significant positive correlations between OD increase of chlorophyll a chlorophyll c and protein concentration at different temperatures. The Fv/Fm parameter achieved a maximum level on the 6(th) or 7(th) day and then decreased to the values registered on the first day of observation. Genetic material undergoes gradual degradation 10 days after inoculation. The size of the cells was invariable.

Zeaxanthin epoxidation - an in vitro approach.

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.

Xanthophyll cycle--a mechanism protecting plants against oxidative stress.

Six different xanthophyll cycles have been described in photosynthetic organisms. All of them protect the photosynthetic apparatus from photodamage caused by light-induced oxidative stress. Overexcitation conditions lead, in the chloroplast, to the over-reduction of the NADP pool and production of superoxide, which can subsequently be metabolized to hydrogen peroxide or a hydroxyl radical, other reactive oxygen species (ROS). On the other hand, overexcitation of photosystems leads to an increased lifetime of the chlorophyll excited state, increasing the probability of chlorophyll triplet formation which reacts with triplet oxygen forming single oxygen, another ROS. The products of the light-dependent phase of xanthophyll cycles play an important role in the protection against oxidative stress generated not only by an excess of light but also by other ROS-generating factors such as drought, chilling, heat, senescence, or salinity stress. Four, mainly hypothetical, mechanisms explaining the protective role of xanthophyll cycles in oxidative stress are presented. One of them is the direct quenching of overexcitation by products of the light phase of xanthophyll cycles and three others are based on the indirect participation of xanthophyll cycle carotenoids in the process of photoprotection. They include: (1) indirect quenching of overexcitation by aggregation-dependent light-harvesting complexes (LHCII) quenching; (2) light-driven mechanisms in LHCII; and (3) a model based on charge transfer quenching between Chl a and Zx. Moreover, results of the studies on the antioxidant properties of xanthophyll cycle pigments in model systems are also presented.