真眼点藻纲的系统分类、生物学特性及应用研究

真眼点藻纲是1970年、1971年Hibberd 和 Leedale根据其细胞学和超微结构的特征, 将原黄藻纲中的一些成员重新调整而成立的一个新纲。自该纲成立以来, 由于新种不断被发现, 科、属和种的数量都有所增加, 目前该纲已有1目6科13个属28个种。该纲藻类的主要细胞光合作用色素包括: 叶绿素a、堇菜黄素、无隔藻黄素和-胡萝卜素; 细胞内具有一周生裂叶状的叶绿体或多个盘状的叶绿体, 具一柄状蛋白核或无, 叶绿体内具有三条类囊体为一组的片层, 无环带形的片层, 双层叶绿体膜外有一层叶绿体内质网膜包裹, 它不与核膜相连; 细胞中具有一个相对较大的、近球形的液泡, 其中含有能振动的颗粒物和一个直径在1-3 m的红色球状体; 繁殖方式通常形成2个D形或4个四面体形的似亲孢子, 有时会形成8个或16个的似亲孢子; 大多种类能产生烧瓶状的游动孢子, 游动孢子具有单根两侧排列管状小茸毛的鞭毛（另一根退化）或另具一根为光滑型的鞭毛。通过18S rDNA和rbcL基因序列的分析, 该纲与异鞭藻门其他各纲藻类的亲缘关系得到确定。脂肪酸分析结果发现该纲的藻类皆含有长链多不饱和脂肪酸-二十碳五烯酸。真眼点藻纲的藻类在淡水、海水和土壤表面等环境中都有分布。该纲的拟微绿球藻属中多个种类被广泛应用于轮虫、卤虫及珍贵海产品幼苗的开口饵料, 亦已成为二十碳五烯酸和重要类胡萝卜素的潜在生产藻株。另外, 研究发现该纲的大多数种类富含油脂, 它们已成为微藻生物燃料开发的备选藻种资源。       

ZOOSPOROGENESIS,MORPHOLOGY, ULTRASTRUCTURE,PIGMENT COMPOSITION,AND PHYLOGENETIC POSITION OF TRACHYDISCUS MINUTUS (EUSTIGMATOPHYCEAE,HETEROKONTOPHYTA)1

The traditional order Mischococcales (Xanthophyceae) is polyphyletic with some original members now classified in a separate class, Eustigmatophyceae. However, most mischococcalean species have not yet been studied in detail, raising the possibility that many of them still remain misplaced. We established an algal culture (strain CCALA 838) determined as one such species, Trachydiscus minutus (Bourr.) H. Ettl, and studied the morphology, ultrastructure, life cycle, pigment composition, and phylogeny using the 18S rRNA gene. We discovered a zoosporic part of the life cycle of this alga. Zoospore production was induced by darkness, suppressed by light, and was temperature dependent. The zoospores possessed one flagellum covered with mastigonemes and exhibited a basal swelling, but a stigma was missing. Ultrastructural investigations of vegetative cells revealed plastids lacking both a connection to the nuclear envelope and a girdle lamella. Moreover, we described biogenesis of oil bodies on the ultrastructural level. Photosynthetic pigments of T. minutus included as the major carotenoids violaxanthin and vaucheriaxanthin (ester); we detected no chl c. An 18S rRNA gene‐based phylogenetic analysis placed T. minutus in a clade with species of the genus Pseudostaurastrum and with Goniochloris sculpta Geitler, which form a sister branch to initially studied Eustigmatophyceae. In summary, our results are inconsistent with classifying T. minutus as a xanthophycean and indicate that it is a member of a novel deep lineage of the class Eustigmatophyceae.     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

PIGMENT AND CYTOLOGICAL EVIDENCE FOR RECLASSIFICATION OF NANNOCHLORIS OCULATA AND MONALIANTUS SALINA IN THE EUSTIGMATOPHYCEAE1

Nannochloris oculata Droop and Monallantus salina Bourrelly produce similar chloroplast pigments and show alike ultrastructural features separating them from the Chlorophyceae and Xanthophyceae, respectively, and linking them to the Eustigmatophyceae. It is proposed that N. oculata should be removed from the chlorophyceaen genus Nannochloris and transferred to a new taxon of coccoid eustigmatophytes containing the closely related M. salina: nomenclatural revisions are required for both strains.     

He-Ne激光和增强UV-B辐射对小麦幼苗类囊体捕光色素的影响

采用5mW.mm-2He-Ne激光辐照、10.08kJ.m-2d-1UV-B辐射及二者组合对冬小麦幼苗进行处理。通过测定叶绿体捕光色素含量和色素蛋白组成的变化,进一步探讨He-Ne激光对增强UV-B辐射后小麦幼苗类囊体捕光色素损伤的修复效应。循环处理小麦幼苗4d,利用90%乙醇和80%丙酮分别提取各处理组小麦幼苗叶片中的叶绿素,通过纸层析和分光光度法检测捕光色素含量的变化,并探讨不同处理对叶绿素与蛋白质结合牢固性的影响。利用柱层析法测定色素蛋白的主要成分。研究表明:与对照组相比,增强UV-B辐照后小麦幼苗捕光色素总含量降低了17.76%,叶绿素和蛋白质结合牢固度显著降低,色素蛋白的组成也发生变化,D1和D2蛋白质条带消失;而一定剂量He-Ne激光辐照可使增强UV-B辐射后的叶绿体色素含量增加约10.64%,但仍低于ck组约8.12%,叶绿素和蛋白质结合牢固度也显著高于B组,色素蛋白的组成与对照组相似。因此,低剂量的He-Ne激光辐照对增强UV-B辐射后小麦幼苗类囊体捕光色素的损伤具有促进修复效应。     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 1987¹, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory,and examples describing applications of the method are provided.     

ISOLATION AND CHARACTERIZATION OF NATIVE PIGMENT-PROTEIN COMPLEXES FROM TWO EUSTIGMATOPHYCEAE1

Pigment-protein complexes were isolated from two species of Eustigmatophyceae, Monodus subterraneus Peterson and Vischeria punctata Vischer, by digitonin treatment followed by density gradient centrifugation. Absorption and fluorescence spectra of the samples were monitored at various steps of preparation, and pigment composition was analyzed by reverse phase HPLC. Although the fluorescence emission spectra were very different in the two species, the absorption spectra were similar, and each exhibited an absorption band with a maximum at 487 nm attributable to violaxanthin and vaucheriaxanthin ester (the molar concentration of these pigments in Monodus was, respectively, 28 and 10 per 100 Chl a). The light-harvesting role of these xanthophylls was ascertained by fluorescence excitation spectra. The light-harvesting fractions (LH) collected in the upper part of the gradient were depleted in β-carotene, whereas their xanthophyll/chlorophyll ratio was almost the same as in whole cells. This is consistent with the presence in these algae of large LH antennae and relatively small core antennae in the photosystems. In Monodus, a polypeptide of 23 kDa, immunologically related to the major LH polypeptide of brown algae, constituted the majority of the LH protein moiety.     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

Characterization of the photosynthetic apparatus of the Eustigmatophycean Nannochloropsis gaditana: Evidence of convergent evolution in the supramolecular organization of photosystem I

Nannochloropsis gaditana belongs to Eustigmatophyceae, a class of eukaryotic algae resulting from a secondary endosymbiotic event. Species of this class have been poorly characterized thus far but are now raising increasing interest in the scientific community because of their possible application in biofuel production. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of only chlorophyll a, with violaxanthin and vaucheriaxanthin esters as the most abundant carotenoids. In this study, the photosynthetic apparatus of this species was analyzed by purifying the thylakoids and isolating the different pigment-binding complexes upon mild solubilization. The results from the biochemical and spectroscopic characterization showed that the photosystem II antenna is loosely bound to the reaction center, whereas the association is stronger in photosystem I, with the antenna-reaction center super-complexes surviving purification. Such a supramolecular organization was found to be conserved in photosystem I from several other photosynthetic eukaryotes, even though these taxa are evolutionarily distant. A hypothesis on the possible selective advantage of different associations of the antenna complexes of photosystems I and II is discussed.     

A simple instrument to perform ‘in vivo’ absorption spectra of pigmented cellular organelles

‘In vivo’ microspectroscopy represents an effective and reliable technique to study pigment distribution and even composition. Contrary to traditional extractive techniques, it preserves the integrity of biological structures, without modifying the nature of the pigments. The spectroscopic apparatus described here is very simple and consists of a standard microscope equipped with an interference filter monochromator, a photomultiplier, and two combined pinhole diaphragms. As examples, absorption spectra of an eyespot and a chloroplast of Euglena gracilis are presented.     

真眼点藻类色素的提取与测定方法

分别采用甲醇、乙醇和丙酮3种有机溶剂提取7种真眼点藻的色素,比较3种有机溶剂提取色素的效果,测定3种有机溶剂色素提取液的吸收光谱,利用分光光度法计算藻的叶绿素a和类胡萝卜素的含量,并比较甲醇和乙醇色素提取液在A₄₇₀和A₆₆₆的最大吸收峰。结果表明:使用乙醇比甲醇和90%丙酮操作更简便、快捷并且毒害低。3种有机溶剂色素提取液的叶绿素a和类胡萝卜素的含量均无显著性差异(P>0.05),提取率基本一致。色素在3种有机溶剂中的吸收光谱相似,甲醇和乙醇的色素提取液在A₄₇₀和A₆₆₆的最大吸收峰并无显著性差异(P>0.05)。乙醇色素提取液可使用Lichtenthaler的公式计算色素含量。     

Variation of leaf traits and pigment content in three species of steppe plants depending on the climate aridity

Mesophyll structure and content of photosynthetic pigments in the leaves of three species of steppe plants, Centaurea scabiosa L., Euphorbia virgata Waldst. et Kit., Helichrysum arenarium (L.) Moench, were investigated in four geographical sites of the Volga region and the Urals located in the forest-steppe and steppe zones. Variations of the studied parameters between geographical points depended both on the species and on the structural organization of the leaf. The highest level of variation was observed for leaf area and pigment content per unit leaf area, the size and the number of chloroplasts in the cell changed to a lesser extent. The leaf thickness, leaf area and mesophyll cell sizes mostly depended on the plant species. C. scabiosa had large leaves (40–50 cm²) with large thickness (280–290 μm) and large mesophyll cells (up to 15000 μm³). The leaves of H. arenarium and E. virgata were ten times smaller and characterized by 1.5 times smaller thickness and 2?3 times smaller cell size. Geographical location and climate of the region affected leaf density, proportion of partial tissue volume, and the ratio of the photosynthetic pigments. In the southern point of Volga region with the highest climate aridity, all studied species were characterized by maximum values of volumetric leaf density (LD), due to the high proportion of sclerenchyma and vascular bundles, and specificity of the mesophyll structure. With the decline in latitude, chlorophyll (Chl) and carotenoid (Car) contents in leaf area were reduced, the ratio Chl/Car was increased, and the ratio Chl a/b was declined. The reduction of the pigment content in the leaf in all species was associated with a reduction in the amount of Chl per chloroplast, and for C. scabiosa and H. arenarium it was associated also with the reduction of chloroplast amount in the leaf area. In turn, chloroplast number per leaf area and the total cell area (A_mes/A) depended on the ratio of the number and size of mesophyll cells inherent to this plant species. At the same time, we found a similar mechanism of spatial organization of leaf restructuring for all studied species—decrease in A_mes/A was accompanied by increasing in the proportion of intercellular air spaces in the leaf. It is concluded that variations in structural and functional parameters of the photosynthetic apparatus of steppe plants were associated with plant adaptation to climate features. General direction of the changes of leaf parameters of the studied species with aridity was the increase of LD and the decrease of pigment content per leaf area however the cellular mechanisms of changes in the pigment content and integral parameters of mesophyll were determined by the plant species properties.     

Carotenoid changes in the peel of ripening persimmon (Diospyros kaki) cv Triumph

The changes of the carotenoid pigments in the peel of ripening persimmon (Diospyros kaki) cv Triumph were followed for an entire season. During ripening, the total carotenoid decreased until colour break, then increased gradually and drastically at the last ripening stages. The chloroplast carotenoid pattern of the unripe fruit changed into a chromoplast pattern in which cryptoxanthin was the predominant pigment, reaching a level between 40 and 50% of the total carotenoids. It accumulated continuously at a rate of approximately 10% at each 2 week interval, its percentage being characteristic for each ripening stage. Other major pigments at levels of approximately 10% of the total carotenoids were zeaxanthin, antheraxanthin and violaxanthin. In the fully ripe fruit, ripened both on and off the tree, lycopene which was not present before was found as the second major pigment. This unusual pattern change is discussed.     

Lutein-chlorophyll-a energy transfer in detergent micelles

Absorption, fluorescence and fluorescence excitation spectra were determined for equimolar mixed micellar detergent solutions of lutein and chlorophyll-a in the concentration range from 9·10^?6 to 1.8·10^?4 M, with detergent (triton-X100) concentrations from 3·-10^?4 to 7·10^?3 M. In the range of detergent concentrations studied the pigments incorporated into the detergent micelles attained a high local concentration (0.1 to 0.01 M), reminiscent of pigment concentration within the chloroplast. A lutein → chlorophyll-a energy transfer with an efficiency of about 15% was found in these systems. In dilute (9·10^?6 M) pigment solution with concentrated (7·10^?3 M) detergent practically no transfer is observed. The extent of aggregation and the efficiency of transfer depend on the composition of the system. The aggregation of chlorophyll-a is partly inhibited by lutein molecules. It is shown that the energy transfer efficiency as function of distance follows anr ^?3 relationship,R ₀ being 22 å.     

Chlorosome antenna complexes from green photosynthetic bacteria

Chlorosomes are the distinguishing light-harvesting antenna complexes that are found in green photosynthetic bacteria. They contain bacteriochlorophyll (BChl) c, d, e in natural organisms, and recently through mutation, BChl f, as their principal light-harvesting pigments. In chlorosomes, these pigments self-assemble into large supramolecular structures that are enclosed inside a lipid monolayer to form an ellipsoid. The pigment assembly is dictated mostly by pigment–pigment interactions as opposed to protein–pigment interactions. On the bottom face of the chlorosome, the CsmA protein aggregates into a paracrystalline baseplate with BChl a, and serves as the interface to the next energy acceptor in the system. The exceptional light-harvesting ability at very low light conditions of chlorosomes has made them an attractive subject of study for both basic and applied science. This review, incorporating recent advancements, considers several important aspects of chlorosomes: pigment biosynthesis, organization of pigments and proteins, spectroscopic properties, and applications to bio-hybrid and bio-inspired devices.     

Glycollate oxidase inhibition and its effect on photosynthesis and pigment formation in Zea mays

We have investigated the effect of 2-hydroxy-3-butynoic acid (HBA) and its methyl ester (MeHBA) on photosynthesis and pigment formation in Zea mays, a C₄ photosynthesis-type plant. In the presence of the specific inhibitor of glycollate oxidase, assimilation of CO₂ was decreased significantly. Labelling patterns showed accumulation of glycollate, though not so marked as in C₃ photosynthesis-type plants, and marked decreases in incorporation into glycine, serine and particularly glycerate. This inhibition was specific for the S(+) enantiomers of HBA and MeHBA. In greening maize R,S-MeHBA inhibited formation of chloroplast pigments and this effect could be shown to be due to the S(+) enantiomer; of a range of metabolises tested only supplementations with serine or pyruvate were partly effective in restoring greening.     

The chloroplast pigments of the algal classes Eustigmatophyceae and Xanthophyceae. II. Xanthophyceae

The chloroplast pigments of Pleurochloris meiringensis Vischer, Mischococcus sphaerocephalus Vischer and Tribonema aequale Pascher have been analysed by a variety of chromatographic methods. There are significant differences between these xanthophycean algae and the eustigmatophycean algae formerly classified with them. In particular the former contain diadinoxanthin as the major xanthophyll whereas diadinoxanthin is absent from the latter and violaxanthin occurs as the major xanthophyll pigment. The other pigments of the Xanthophyceae sensu stricto include chlorophyll a, β-carotene, vaucheriaxanthin-ester, heteroxanthin, diatoxanthin, cryptoxanthin epoxide and a neoxanthin-like pigment.     

Profiles of photosynthetic pigment accumulation and expression of photosynthesis-related genes in the marine cyanobacteria Synechococcus sp.: Effects of LED wavelengths

Light quality is a significant environmental factor that influences photosynthetic pigments in cyanobacteria. In the present study, we illuminated the marine cyanobacteria Synechococcus sp. with white (350 ～ 700 nm), red (630 nm), green (530 nm), and blue (450 nm) light emitting diodes (LEDs) and measured pigment levels (chlorophyll, carotenoid, and phycobiliprotein) and expression of photosynthesis-related genes (pebA, psbB, and psaE). The amount of photosynthetic pigments (total pigments, chlorophyll, and phycobiliproteins) was higher in the green and blue LED groups than in the white and red LED groups after 8 days of culture. The cells were prepared in a 1.5 mL solution for the analysis of the total pigments, chlorophyll, and carotenoid, and in a 2 mL for analysis of phycobiliproteins. The mRNA expression levels of pebA and psbB significantly increased after 8 days of cultivation under green and blue light, while the mRNA expression levels of psaE decreased. These results indicate that green and blue light increase the accumulation of photosynthetic pigments. In contrast red light induced mRNA expression of psaE and stimulated cell growth in Synechococcus sp.     

Pigment Production on L-Tryptophan Medium by Cryptococcus gattii and Cryptococcus neoformans

In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.