Pigment composition,optical properties,and resistance to photodamage of the microalga <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> cultivated under high light

The relationships between changes in cell suspension absorbance, pigment composition, and resistance to photodamage were investigated in the microalga Haematococcus pluvialis Flotow em. Wille (Chlorophyta) IPPAS H-239 cultivated under high level of photosynthetically active radiation (PAR, 50 W/m²). When the green flagellated cells of H. pluvialis lacking astaxanthin (Ast) and possessing low (<0.5) carotenoid/chlorophyll ratio were irradiated by intense light (2500 W/m² PAR), rapid and synchronous photobleaching of 70–80% of chlorophyll (Chl) and carotenoids (Car) was observed. By contrast, the rate of pigment photobleaching in cells with Car/Chl > 1, which retained high Chl content (> 0.6 fmol/cell) and accumulated significant amounts of Ast, was two times lower than in the green cells. Red aplanospores, with Car/Chl > 10, containing high amounts of Ast and low amounts of Chl (> 0.8 and < 0.1 fmol/cell, respectively) were resistant to photodestruction. The extent of cell resistance to photobleaching correlated closely with an increase in contribution of Car to light absorption by H. pluvialis cell suspensions. The build up of Ast during acclimation to high light was accompanied by a gradual increase in the optical density ratio OD₄₈₀/OD₆₇₈, whereas synchronous (OD₄₈₀/OD₆₇₈ ≈ const; r ² > 0.99) and profound (>20%) bleaching of Car and Chl absorption bands was characteristic of photodamage. The spectral features of photoacclimation and photodamage revealed in this work can be used for nondestructive diagnostics of photodamage in H. pluvialis cultures and for on-line assessment of cell resistance to photooxidative death. The results are discussed with respect to the nondestructive monitoring of laboratory and production cultures of H. pluvialis and their protection from photooxidative death.     

A Spectrophotometric Analysis of Pigments in Apples

Methods of pigment extraction using traditional polar organic solvents (acetone or methanol) were compared to those employing a chloroform–methanol mixture. We found that, for spectrophotometric pigment analysis in the apple peel, the cuticular lipids must be preliminarily extracted from the samples with chloroform and MgO must be added during homogenization to prevent pigment degradation. The traditional extraction did not result in the complete extraction of intact pigments, and such extracts contained a considerable amount of light-absorbing impurities. The application of chloroform–methanol extraction allowed us to markedly reduce the content of such impurities and to increase the accuracy and sensitivity of the measurement of the content of chlorophylls and carotenoids. In addition, this extraction method proved useful for the analysis of phenolic substances (anthocyanins and flavonoids) in the water-methanol fraction of the extracts.     

Tolerance of the photosynthetic apparatus to acidification of the growth medium as a possible determinant of CO<Subscript>2</Subscript>-tolerance of the symbiotic microalga <Emphasis Type="Italic">Desmodesmus</Emphasis> sp. IPPAS-2014

The symbiotic unicellular chlorophyte Desmodesmus sp. IPPAS-2014 capable of growth at extremely high CO₂ levels prohibitive for most other microalgae is an interesting model for studies of CO₂ tolerance mechanisms and a promising organism for CO₂ biocapture. We studied the initial (0-60 min) phase of acclimation of this microalga to an abrupt decrease in pH of the medium sparged with air/20% CO₂ mixture. Acclimation of the culture to these conditions was accompanied by a sharp decrease in photochemical activity of the chloroplast followed by its recovery with a characteristic time of 10-50 min. We hypothesize that acidification of the cultivation medium by dissolving CO₂ plays a key role in the observed decrease in the photochemical activity. The possible role of photosynthetic apparatus tolerance to abrupt acidification in overall high tolerance of symbiotic microalgae to extremely high CO₂ levels is discussed.     

An exceptional irradiance-induced decrease of light trapping in two Tradescantia species: an unexpected relationship with the leaf architecture and zeaxanthin-mediated photoprotection

Leaf anatomy and irradiance-dependent leaf transmittance changes serving as irradiance acclimation mechanisms in leaves were studied in two ecologically contrasting Tradescantia species, a shade plant T. fluminensis Vell. and a sun plant T. sillamontana Matuda, grown at different irradiances. A dramatic increase in leaf thickness (2 to 4-fold) under a high growth irradiance (800 μmol m^?2 s^?1) compared with a low growth irradiance (60 μmol m^?2 s^?1), achieved mainly by expansion of the epidermis, was recorded in both species. The effect took place on the background of modest changes in mesophyll thickness (1.8-fold in T. fluminensis and 1.15-fold in T. sillamontana) and chloroplast size (0.8-fold in T. fluminensis and an insignificant change in T. sillamontana). Mesophyll structure and growth irradiance response did not seem to facilitate significantly light-dependent chloroplast (avoidance) movement in these species. Nevertheless, an exceptionally large (2 to 4-fold) irradiance-induced increase in light transmittance attributable to chloroplast avoidance movement was revealed. This increase by far exceeded that in other higher plants according to available literature. The magnitude of the irradiance-dependent transmittance changes positively correlated both with the rate of photosystem II recovery and with the extent of xanthophyll deepoxidation in the leaves. This was opposite to a negative correlation observed between the same parameters in different plant species. We hypothesize that, at the evolutionary timescale, chloroplast avoidance movement might adjust independently from other photoprotective mechanisms, e.g., non-photochemical quenching, whereas, on the ontogenetic timescale, adjustment of these mechanisms inevitably follows the same trend.     

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.     

Stress-Induced Changes in Optical Properties,Pigment and Fatty Acid Content of <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.: Implications for Non-destructive Assay of Total Fatty Acids

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m² s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m² s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass–fiber filters in the chlorophyll content range of 3–13 mg/L. Under stressful conditions, a 30–50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15–45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3–0.6; root mean square error (RMSE) = 0.03; r ² = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0–45%; RMSE = 3.23 %; r ² = 0.89) in the broad band 400–550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.     

EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES,PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A₄₇₆/A₆₇₆ and A₆₅₀/A_676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.     

Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incisa

The effects of light and nitrogen deficiency on biomass, fatty acid content and composition were studied in Parietochloris incisa, the unicellular freshwater chlorophyte accumulating very high amounts of arachidonic-acid-rich triacylglycerols. P. incisa cultures grown on complete nutrient medium and under high light (400 μmol photons m^{− 2} s⁻¹) showed the highest rate of growth in comparison to medium (200 μmol photons m⁻² s⁻¹) and low (35 μmol photons m⁻² s⁻¹) light intensity. Cultures grown under high light (on complete BG-11 medium) attained higher volumetric contents of total fatty acids and arachidonic acid due to greater increase in biomass. Nitrogen starvation brought about a strong increase in the arachidonic acid proportion of total fatty acids. Thus, adjustments to cultivation conditions could serve as an efficient tool for manipulation of yield and relative content of arachidonic acid in P. incisa. The significance of the changes in lipid metabolism for adaptation of P. incisa to high-light stress and nitrogen deficiency is also discussed.     

Versatility of the green microalga cell vacuole function as revealed by analytical transmission electron microscopy

Vacuole is a multifunctional compartment central to a large number of functions (storage, catabolism, maintenance of the cell homeostasis) in oxygenic phototrophs including microalgae. Still, microalgal cell vacuole is much less studied than that of higher plants although knowledge of the vacuolar structure and function is essential for understanding physiology of nutrition and stress tolerance of microalgae. Here, we combined the advanced analytical and conventional transmission electron microscopy methods to obtain semi-quantitative, spatially resolved at the subcellular level information on elemental composition of the cell vacuoles in several free-living and symbiotic chlorophytes. We obtained a detailed record of the changes in cell and vacuolar ultrastructure in response to environmental stimuli under diverse conditions. We suggested that the vacuolar inclusions could be divided into responsible for storage of phosphorus (mainly in form of polyphosphate) and those accommodating non-protein nitrogen (presumably polyamine) reserves, respectively.The ultrastructural findings, together with the data on elemental composition of different cell compartments, allowed us to speculate on the role of the vacuolar membrane in the biosynthesis and sequestration of polyphosphate. We also describe the ultrastructural evidence of possible involvement of the tonoplast in the membrane lipid turnover and exchange of energy and metabolites between chloroplasts and mitochondria. These processes might play a significant role in acclimation in different stresses including nitrogen starvation and extremely high level of CO₂ and might also be of importance for microalgal biotechnology. Advantages and limitations of application of analytical electron microscopy to biosamples such as microalgal cells are discussed.