Effect of Abscisic Acid on Growth, Fatty Acid Profile, and Pigment Composition of the Chlorophyte Chlorella (Chromochloris) zofingiensis and Its Co-Culture Microbiome.

Microalga Chlorella (Chromochloris) zofingiensis has been gaining increasing attention of investigators as a potential competitor to Haematococcus pluvialis for astaxanthin and other xanthophylls production. Phytohormones, including abscisic acid (ABA), at concentrations relevant to that in hydroponic wastewater, have proven themselves as strong inductors of microalgae biomass productivity and biosynthesis of valuable molecules. The main goal of this research was to evaluate the influence of phytohormone ABA on the physiology of C. zofingiensis in a non-aseptic batch experiment. Exogenous ABA stimulated C. zofingiensis cell division, biomass production, as well as chlorophyll, carotenoid, and lipid biosynthesis. The relationship between exogenous ABA concentration and the magnitude of the observed effects was non-linear, with the exception of cell growth and biomass production. Fatty acid accumulation and composition depended on the concentration of ABA tested. Exogenous ABA induced spectacular changes in the major components of the culture microbiome of C. zofingiensis. Thus, the abundance of the representatives of the genus Rhodococcus increased drastically with an increase in ABA concentration, whereas the abundance of the representatives of Reyranella and Bradyrhizobium genera declined. The possibilities of exogenous ABA applications for the enhancing of the biomass, carotenoid, and fatty acid productivity of the C. zofingiensis cultures are discussed.

Assessment of the Tolerance of a Chlorophyte Desmodesmus to CuO-NP for Evaluation of the Nanopollution Bioremediation Potential of This Microalga.

Broad application of CuO nanoparticles (CuO-NP) for industrial and household purposes leads to a continuous increase in their discharge to, and, hence, ever-increasing environmental hazards for aquatic ecosystems. Microalgae-based technologies hold promise for bioremediation of diverse hazardous micropollutants (HMP), including NP, from wastewater. In this study, we tested the ability of the green microalga Desmodesmus sp. to accumulate CuO-NP or their components. We also assessed the tolerance of this microalga to the environmentally relevant concentrations of CuO-NP. Using scanning electron microscopy, we demonstrated that the average size of CuO-NP was 50-100 nm, and their purity was confirmed with elemental composition analysis. Tests of the colloidal suspensions of CuO-NP showed that the hydrodynamic diameter of CuO-NP and their aggregates was below 100 nm. Flow cytometry analysis showed that CuO-NP at a concentration of 100 µg L-1 slightly inhibited the viability of microalgae cells and led to an increase in their oxidative stress. The assessment of the condition of photosystem II showed that CuO-NP exert a multifaceted effect on the photosynthetic apparatus of Desmodesmus sp., depending on the concentration of and the exposure to the CuO-NP. Desmodesmus sp. turned to be relatively tolerant to CuO-NP. In addition, the ICP-MS method revealed increased bioaccumulation of copper by microalgae cells in the experimental groups. The outcomes of this study indicate that the Desmodesmus sp. has a significant potential for bioremoval of the copper-based nanostructured HMP from an aquatic environment.

Chilling Upregulates Expression of the PsbS and LhcSR Genes in the Chloroplasts of the Green Microalga Lobosphaera incisa IPPAS C-2047.

Non-photochemical quenching (NPQ) of excited chlorophyll states is essential for protecting the photosynthetic apparatus (PSA) from the excessive light-induced damage in all groups of oxygenic photosynthetic organisms. The key component of the NPQ mechanism in green algae and some other groups of algae and mosses is the LhcSR protein of the light harvesting complex (LHC) protein superfamily. In vascular plants, LhcSR is replaced by PsbS, another member of the LHC superfamily and a subunit of photosystem II (PSII). PsbS also performs the photoprotective function in mosses. For a long time, PsbS had been believed to be nonfunctional in green algae, although the corresponding gene was discovered in the genome of these organisms. The first evidence of the PsbS accumulation in the model green alga Chlamydomonas reinhardtii in response to the increase in irradiance was obtained only six years ago. However, the observed increase in the PsbS content was short-termed (on an hour-timescale). Here, we report a significant (more than three orders of magnitude) and prolonged (four days) upregulation of PsbS expression in response to the chilling-induced high-light stress followed by a less significant (~ tenfold) increase in the PsbS expression for nine days. This is the first evidence for the long-term upregulation of the PsbS expression in green alga (Chlorophyta) in response to stress. Our data indicate that the role of PsbS in the PSA of Chlorophyta is not limited to the first-line defense against stress, as it was previously assumed, but includes full-scale participation in the photoprotection of PSA from the environmental stress factors.

The Effect of Chilling on the Photosynthetic Apparatus of Microalga Lobosphaera incisa IPPAS C-2047.

Photosynthetic organisms have developed a set of mechanisms aimed at preventing photo-oxidative reactions in the photosynthetic apparatus (PSA) initiated by excessively absorbed light energy. Along with high irradiance, other stressors, e.g., chilling temperatures, can lead to the absorption of the excess of light energy and hence to photo-oxidative stress. Here, we studied induction of photoprotective mechanisms in response to chilling (0°C) at a low irradiance (50 µmol PAR photons m-2·s-1) in the cells of microalga Lobosphaera incisa IPPAS C-2047. After 4 days of incubation at a low temperature, L. incisa IPPAS C-2047 cells showed a notable decrease in the photochemical activity of photosystem II (PSII) and in the efficiency of photosynthetic electron transport, as well as a significant increase in the thermal dissipation of the absorbed light energy in the light-harvesting antenna. In contrast, most conventional markers of PSA acclimation to excess light energy [total chlorophyll and carotenoid content; violaxanthin cycle pigment content and de-epoxidation state; photosynthetic antenna, PSII, and photosystem I (PSI) ratio] remained virtually unchanged. The content of major unsaturated fatty acids also remained almost unaffected, except for arachidonic acid (increased by 40%) recently assumed to activate violaxanthin de-epoxidase by adjusting its lipid microenvironment. Significant changes (4-7-fold increase) were observed in the expression of the gene encoding protective protein LhcSR. Pre-conditioning at 5°C prior to the acclimation to 0°C augmented the PSA photochemical activity. Our data show that the mid-term (4-d) acclimation of L. incisa IPPAS C-2047 to a chilling temperature at a low irradiance triggers the PSA response resembling, in part, the response to high light but relying mostly on the LhcSR protein-dependent quenching of excitation in the photosynthetic antenna.

Simulating the Interplay between the Uptake of Inorganic Phosphate and the Cell Phosphate Metabolism under Phosphorus Feast and Famine Conditions in Chlorella vulgaris.

Using a mathematical simulation approach, we studied the dynamics of the green microalga Chlorella vulgaris phosphate metabolism response to shortage and subsequent replenishing of inorganic phosphate in the medium. A three-pool interaction model was used to describe the phosphate uptake from the medium, its incorporation into the cell organic compounds, its storage in the form of polyphosphates, and culture growth. The model comprises a system of ordinary differential equations. The distribution of phosphorous between cell pools was examined for three different stages of the experiment: growth in phosphate-rich medium, incubation in phosphate-free medium, and phosphate addition to the phosphorus-starving culture. Mathematical modeling offers two possible scenarios for the appearance of the peak of polyphosphates (PolyP). The first scenario explains the accumulation of PolyP by activation of the processes of its synthesis, and the decline in PolyP is due to its redistribution between dividing cells during growth. The second scenario includes a hysteretic mechanism for the regulation of PolyP hydrolysis, depending on the intracellular content of inorganic phosphate. The new model of the dynamics of P pools in the cell allows one to better understand the phenomena taking place during P starvation and re-feeding of the P-starved microalgal cultures with inorganic phosphate such as transient PolyP accumulation. Biotechnological implications of the observed dynamics of the polyphosphate pool of the microalgal cell are considered. An approach enhancing the microalgae-based wastewater treatment method based on these scenarios is proposed.

Spectrum of Light as a Determinant of Plant Functioning: A Historical Perspective.

The significance of the spectral composition of light for growth and other physiological functions of plants moved to the focus of "plant science" soon after the discovery of photosynthesis, if not earlier. The research in this field recently intensified due to the explosive development of computer-controlled systems for artificial illumination and documenting photosynthetic activity. The progress is also substantiated by recent insights into the molecular mechanisms of photo-regulation of assorted physiological functions in plants mediated by photoreceptors and other pigment systems. The spectral balance of solar radiation can vary significantly, affecting the functioning and development of plants. Its effects are evident on the macroscale (e.g., in individual plants growing under the forest canopy) as well as on the meso- or microscale (e.g., mutual shading of leaf cell layers and chloroplasts). The diversity of the observable effects of light spectrum variation arises through (i) the triggering of different photoreceptors, (ii) the non-uniform efficiency of spectral components in driving photosynthesis, and (iii) a variable depth of penetration of spectral components into the leaf. We depict the effects of these factors using the spectral dependence of chloroplast photorelocation movements interlinked with the changes in light penetration into (light capture by) the leaf and the photosynthetic capacity. In this review, we unfold the history of the research on the photocontrol effects and put it in the broader context of photosynthesis efficiency and photoprotection under stress caused by a high intensity of light.

Cationic penetrating antioxidants switch off Mn cluster of photosystem II in situ.

Mitochondria-targeted antioxidants (also known as 'Skulachev Ions' electrophoretically accumulated by mitochondria) exert anti-ageing and ROS-protecting effects well documented in animal and human cells. However, their effects on chloroplast in photosynthetic cells and corresponding mechanisms are scarcely known. For the first time, we describe a dramatic quenching effect of (10-(6-plastoquinonyl)decyl triphenylphosphonium (SkQ1) on chlorophyll fluorescence, apparently mediated by redox interaction of SkQ1 with Mn cluster in Photosystem II (PSII) of chlorophyte microalga Chlorella vulgaris and disabling the oxygen-evolving complex (OEC). Microalgal cells displayed a vigorous uptake of SkQ1 which internal concentration built up to a very high level. Using optical and EPR spectroscopy, as well as electron donors and in silico molecular simulation techniques, we found that SkQ1 molecule can interact with Mn atoms of the OEC in PSII. This stops water splitting giving rise to potent quencher(s), e.g. oxidized reaction centre of PSII. Other components of the photosynthetic apparatus proved to be mostly intact. This effect of the Skulachev ions might help to develop in vivo models of photosynthetic cells with impaired OEC function but essentially intact otherwise. The observed phenomenon suggests that SkQ1 can be applied to study stress-induced damages to OEC in photosynthetic organisms.

Express Analysis of Microalgal Secondary Carotenoids by TLC and UV-Vis Spectroscopy.

We elucidate the peculiarities of express analysis of secondary carotenoids from microalgae and their preliminary identification using TLC and UV-Vis spectroscopy with emphasis on biotechnologically significant producers of the value-added pigment astaxanthin. Advantages and limitations of the method are described to underline the value of TLC as a potential companion method to mainstream separation techniques such as HPLC. Special attention is paid to common errors and pitfalls of the method and possible work-arounds, as well as to overall strategy of the analysis, sample preparation, and material selection.

Acclimation of shade-tolerant and light-resistant Tradescantia species to growth light: chlorophyll a fluorescence, electron transport, and xanthophyll content.

In this study, we have compared the photosynthetic characteristics of two contrasting species of Tradescantia plants, T. fluminensis (shade-tolerant species), and T. sillamontana (light-resistant species), grown under the low light (LL, 50-125 µmol photons m-2 s-1) or high light (HL, 875-1000 µmol photons m-2 s-1) conditions during their entire growth period. For monitoring the functional state of photosynthetic apparatus (PSA), we measured chlorophyll (Chl) a emission fluorescence spectra and kinetics of light-induced changes in the heights of fluorescence peaks at 685 and 740 nm (F 685 and F 740). We also compared the light-induced oxidation of P700 and assayed the composition of carotenoids in Tradescantia leaves grown under the LL and HL conditions. The analyses of slow induction of Chl a fluorescence (SIF) uncovered different traits in the LL- and HL-grown plants of ecologically contrasting Tradescantia species, which may have potential ecophysiological significance with respect to their tolerance to HL stress. The fluorometry and EPR studies of induction events in chloroplasts in situ demonstrated that acclimation of both Tradescantia species to HL conditions promoted faster responses of their PSA as compared to LL-grown plants. Acclimation of both species to HL also caused marked changes in the leaf anatomy and carotenoid composition (an increase in Violaxanthin + Antheraxantin + Zeaxanthin and Lutein pools), suggesting enhanced photoprotective capacity of the carotenoids in the plants grown in nature under high irradiance. Collectively, the results of the present work suggest that the mechanisms of long-term PSA photoprotection in Tradescantia are based predominantly on the light-induced remodeling of pigment-protein complexes in chloroplasts.

Recent breakthroughs in the biology of astaxanthin accumulation by microalgal cell.

Massive accumulation of the secondary ketokarotenoid astaxanthin is a characteristic stress response of certain microalgal species with Haematococcus pluvialis as an illustrious example. The carotenogenic response confers these organisms a remarkable ability to survive in extremely unfavorable environments and makes them the richest source of natural astaxanthin. Exerting a plethora of beneficial effects on human and animal health, astaxanthin is among the most important bioproducts from microalgae. Though our understanding of astaxanthin biosynthesis, induction, and regulation is far from complete, this gap is filling rapidly with new knowledge generated predominantly by application of advanced "omics" approaches. This review focuses on the most recent progress in the biology of astaxanthin accumulation in microalgae including the genomic, proteomic, and metabolomics insights into the induction and regulation of secondary carotenogenesis and its role in stress tolerance of the photosynthetic microorganisms. Special attention is paid to the coupling of the carotenoid and lipid biosynthesis as well as deposition of astaxanthin in the algal cell. The place of the carotenogenic response among the stress tolerance mechanisms is revisited, and possible implications of the new findings for biotechnological production of astaxanthin from microalgae are considered. The potential use of the carotenogenic microalgae as a source not only of value-added carotenoids, but also of biofuel precursors is discussed.

Similarity and diversity of the Desmodesmus spp. microalgae isolated from associations with White Sea invertebrates.

Similarity and diversity of the phenotype and nucleotide sequences of certain genome loci among the single-celled microalgae isolated from White Sea benthic invertebrates were studied to extend the knowledge of oxygenic photoautotrophs forming microbial communities associated with animals. We compared four Desmodesmus isolates (1Hp86E-2, 1Pm66B, 3Dp86E-1, 2Cl66E) from the sponge Halichondria panicea, trochophore larvae of the polychaete Phyllodoce maculata, and the hydroids Dynamena pumila and Coryne lovenii, respectively. The microalgae appeared to be very similar featuring the phenotypic and genetic traits characteristics of unicellular representatives of the genus Desmodesmus. At the same time, isolates from different animal species displayed certain differences in (i) the epistructure morphology; (ii) type and number of the inclusions such as interthylakoid starch grains and cytoplasmic oil bodies and (iii) fatty acid composition; in Desmodesmus sp. 1Hp86E-2, these differences were most pronounced. Phylogenetic analysis based on ITS1-5.8S rRNA-ITS2 and rbcL sequences showed that all isolates studied differ from known classified representatives of Desmodesmus combining a deletion in the conservative 5.8S rRNA gene and long AC-microsatellite repeats in the ITS1 whereas 1Hp86E-2 represented a distinct branch within this group.

Light absorption by anthocyanins in juvenile, stressed, and senescing leaves.

The optical properties of leaves from five species, Norway maple (Acer platanoides L.), cotoneaster (Cotoneaster alaunica Golite), hazel (Corylus avellana L.), Siberian dogwood (Cornus alba L.), and Virginia creeper (Parthenocissus quinquefolia (L.) Planch.), differing in pigment composition and at different stages of ontogenesis, were studied. Anthocyanin absorption maxima in vivo, as estimated with spectrophotometry of intact anthocyanic versus acyanic leaves and microspectrophotometry of vacuoles in the leaf cross-sections, were found between 537 nm and 542 nm, showing a red shift of 5-20 nm compared with the corresponding maxima in acidic water-methanol extracts. In non-senescent leaves, strong anthocyanin absorption was found between 500 nm and 600 nm (with a 70-80 nm apparent bandwidth). By and large, absorption by anthocyanin in leaves followed a modified form of the Lambert-Beer law, showing a linear trend up to a content of nearly 50 nmol cm(-2), and permitting thereby a non-invasive determination of anthocyanin content. The apparent specific absorption coefficients of anthocyanins at 550 nm showed no substantial dependence on the species. Anthocyanin contribution to total light absorption at 550 nm was followed in maple leaves in the course of autumn senescence. Photoprotection by vacuolar anthocyanins is discussed with special regard to their distribution within a leaf; radiation screening by anthocyanins predominantly localized in the epidermal cells in A. platanoides and C. avellana leaves was also evaluated.

Apple flavonols during fruit adaptation to solar radiation: spectral features and technique for non-destructive assessment.

Spectral properties of flavonols of three varieties (Golden Delicious, Antonovka, and Renet Simirenko) of anthocyanin-free apple fruit were investigated with reflectance spectroscopy. The results of spectral and biochemical analyses suggested that fruit reflectance in a broad spectral range 365-430 nm is strongly dependent on and, in sunlit fruit surfaces, governed by flavonols. The build up of peel flavonols (mainly rutin and other quercetin glycosides) resulted in a dramatic decrease of fruit reflectance in this range, flattening of the spectrum, and extending the region with low reflectance (4-5%) to ca. 410 nm. The spectral features observed suggest that flavonols contribute significantly to screening of excessive radiation, not only UV-A, but in the short-wave bands of chlorophyll and carotenoid absorption in the visible part of the spectrum as well. To retrieve quantitatively flavonol content from reflectance spectra, we tested the applicability of an inversion technique developed for non-destructive leaf pigment assessment. The model for flavonol content assessment was suggested in the form (R(-1)410 - R(-1)460)R800, where Rlambda is reflectance at wavelength lambda. The model was linearly related to flavonol content between 8 and 220nmol/cm2 with the coefficient of determination r2=0.92 and root mean square error of flavonol estimation of 20 nmol/ cm2 regardless of cultivar, chlorophyll, and carotenoid content.

Involvement of phytochrome in regulation of transpiration: red-/far red-induced responses in the chlorophyll-deficient mutant of pea.

Transpiration rhythmicity and intensity were investigated in the chlorophyll-deficient mutant XL18 of Pisum sativum L. and in the phytochrome-deficient mutant aurea of Lycopersicon esculentum Mill. A custom-built psychrometer was used. In the XL18 mutant an acute transpiration response to monochromatic irradiation was observed such that red (R) light increased and far-red (FR) decreased transpiration rate, with equal rates of change. This result indicates that phytochrome is involved in regulation of transpiration. In wild type pea the chlorophyll-dependent component of transpiration was also shown to involve phytochrome. Monochromatic irradiation by red or far-red light induced an increase in transpiration with acceleration dependent on time of day. The response was irreversible by light of either wavelength. We conclude that both photoreceptors are involved in the acute response. Investigation of the daily course of transpiration revealed rhythmic changes in wild type pea and tomato under natural light conditions and in constant darkness. The rhythm was not apparent in the XL18 mutant in constant darkness, or in the aurea mutant under natural illumination. The latter results show that phytochrome, as a photoreceptor, is essential for maintaining the rhythm upon irradiation, while the photosynthetic component is crucial in darkness.