Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

After first being analyzed by HPLC, 4 free carotenoids, 15 astaxanthin monoesters, 12 astaxanthin diesters, and 3 astacin monoesters in Haematococcus pluvialis were identified by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-(APCI)MS). Identification of each compound was based on the characteristic fragment ions of the positive ion mode, negative ion mode, and MS(2). Astaxanthin esters were identified based on the loss of one or two fatty acids. In a positive ion mode, astaxanthin monoesters had characteristic fragment ions at m/z 597 [M+H-fatty acid](+) and m/z 579 and 561 that resulted from a continuous loss of water. The relative intensity of m/z 579 in MS(2) amounted to more than 80% of that of the molecular ion. In astaxanthin diesters, the intensity of m/z 561 occasionally was equal to that of m/z 579, but in general the former, amounting to 50 to 60% or more of the molecular ion, was stronger than the latter, which decreased to 20 to 30% of the molecular ion. In addition, a set of compounds with maximum absorbance at 400 nm, detected by high-performance liquid chromatography-diode array detector (HPLC-DAD), had strong characteristic fragment ions at m/z 871 and 593 in the positive ion mode MS(2). They were presumed to be linolenic acid or an isomer of omega-6-gamma-linolenic acid esters of astacin.     

Determination of an ethylene biosynthesis pathway in the unicellular green alga,Haematococcus pluvialis. Relationship between growth and ethylene production

In the freshwater ChlorophyceaeHaematococcus pluvialis, precursors of ethylene biosynthesis cycle are the same as those of higher plants: L-methionine S-adenosylmethionine 1-aminocyclopropane-1-carboxylic acid ethylene. However, the enzymatic complex of the last step of ethylene synthesis-ACCoxidase-differs from that of higher plants. It is stimulated by Co²⁺ (at least 10^-5 M), Mn²⁺ (at least 10^-6 M) and Ag²⁺ (at least 10^-4 M), inhibited by Cu²⁺ (at least 10^-5 M) and not affected by Zn²⁺, Fe²⁺ or Mg²⁺. ACCoxidase is also inhibited by salicylhydroxamic acid and by dark. Ethylene production is more important in young, mobile, green cells in active growth phase than in old, encysted and red cells in stationary growth phase. No peaks in ethylene production or respiration were observed during batch culture, as opposed to the situation with climacteric fruits.     

Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N-starvation and high light conditions

Haematococcus pluvialis gave the highest astaxanthin accumulation rate (2.7 mg l^–1 day^–1) and total astaxanthin content ( 22.7 mg g^–1 biomass). Astaxanthin accumulation in Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Chorella zofingiensis and Scenedesmus vacuolatus was, respectively, 19.2, 14.3, 10.9, 6.8 and 2.7 mg astaxanthin g^–1 biomass, respectively.     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

Accumulation and protection activity of protease-resistant heat-stable proteins in Haematococcus pluvialis during high light and nitrogen starvation

Under stress conditions of high light andnitrogen starvation the green motile cellsof the unicellular green algaHaematococcus pluvialis are known tocease growing and transform into inert redcysts, in which the secondary carotenoidastaxanthin accumulates. A study wastherefore made on other effects of suchconditions. A number ofprotease-resistant, heat-stable proteinswith apparent molecular masses of 38 kDa,50 kDa, 62 kDa and 63 kDa accumulated. Thisprotein fraction was effective in theprotection of horseradish peroxidase frominactivation, suggesting a role for theseproteins in H. pluvialis subjected toa stress event.     

Characterization of a New Stearoyl-acyl Carrier Protein Desaturase Gene from Jatropha curcas

A new full-length cDNA of stearoyl-acyl carrier protein desaturase was obtained by RT-PCR and RACE techniques from developing seeds of Jatropha curcas. Sequence alignment showed that its deduced amino acid sequence had high similarity with other stearoyl-acyl carrier protein desaturases. The gene was functionally expressed in E. coli and the desaturating activity of recombinant protein was easily detected when assayed in vitro with added spinach ferredoxin. Southern blot analysis indicated that the gene was a member of a small gene family. Northern blot analysis revealed it was highly expressed in developing fruits of J. curcas. Revisions requested 16 December 2005; Revisions received 6 February 2006     

Interplay between photochemical activities and pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to red stage

The transfer of laboratory cultures of H. pluvialis to high irradiance outdoors caused a substantial decline in the maximum quantum yield of photosystem II (PSII), from 0.65 in the morning to 0.45 at midday, as measured by the ratio of variable to maximum fluorescence yields (F_v/F_m), and a steep rise in non-photochemical quenching (NPQ). Chlorophyll fluorescence induction curves of morning samples showed a clear I-step, reflecting a certain PSII heterogeneity. Single turnover flash measurements on samples taken from the outdoor photobioreactor in the middle of day showed an increase in the reoxidation time constant of the reduced plastoquinone Q_A ^–, i.e., the time required for electron transfer from the primary plastoquinone acceptor of PSII Q_A ^– to the secondary plastoquinone acceptor Q_B. Photosynthesis rates were almost constant during the day. Along with the increase in non-photochemical quenching, there was a slight increase in zeaxanthin and antheraxanthin contents and decrease in violaxanthin, showing the presence of an operative xanthophyll cycle in this microalga. A marked increase of secondary carotenoids was found at the end of the first day of exposure to sunlight, mainly astaxanthin monoester, which reached 15.5% of the total carotenoid content. Though cells turned reddish during the second day, the decline in the fluorescence parameter F_v/F_m in the middle of the day was less than during the first day, and there was no further increase in the value for NPQ. Similar behaviour was observed during the third day when the culture was fully red. After four days of exposure to sunlight, the dry weight reached 800 mg L^–1 and the concentration of secondary carotenoids (81% astaxanthin monoester) reached 4.4% dry weight.     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

Cloning and Sequence Analysis of the Gene Encoding (6-4)photolyase from Dunaliella salina

DNA photolyase can repair UV-induced DNA damage in a light-dependent manner. A cDNA of (6-4)photolyase from Dunaliella salina (GenBank accession number: AY845324) was cloned, sequenced and its amino acid sequence was deduced. The derived amino acid sequence showed high homology with other (6-4)photolyases and a predicted 3D model was constructed by homology modeling. Revisions requested 20 May 2005 and 18 August 2005; Revisions received 2 August 2005 and 28 November 2005     

Cloning and Characterization of an Agglutinin Gene from Arisaema lobatum

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5′-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Molecular phylogenetics and biogeography of Neotropical piping guans (Aves: Galliformes): Pipile Bonaparte, 1856 is synonym of Aburria Reichenbach, 1853

The Cracidae are Neotropical galliform birds with 11 genera currently recognized. To investigate the questioned validity of Pipile Bonaparte, 1856 and the monotypic Aburria Reichenbach, 1853 as separate genera, we gathered data from 2727 bp of mitochondrial DNA (cytochrome b, ND2 and control region) and 151 osteological characters. Our phylogenetic analyses of DNA sequences indicated that Aburria aburri is embedded within Pipile. Also, genetic distances between Aburria and any Pipile species are equivalent to the distances estimated for other congeneric cracid species, which genus status is not doubtful. Although the osteological characters do not have phylogenetic signal to solve the phylogenetic relationships at species level, five synapomorphies were found for Aburria and Pipile. Therefore, we suggest that Pipile should be merged with Aburria, which is the oldest described genus. We estimated that speciation in this group occurred in the Plio-Pleistocene, concordant with other birds, primates and rodents that have similar geographic distribution, and proposed a diversification hypothesis based on the occurrence of sea transgressions and the formation of the Amazon Lagoon. Therefore, we conclude that these palaeogeographic events may have contributed to Neotropical taxa diversification to a greater extent than previously suspected.