M.A. Vargas

BIOCHEMICAL COMPOSITION AND FATTY ACID CONTENT OF FILAMENTOUS NITROGEN-FIXING CYANOBACTERIA

 The biochemical composition and fatty acid content of twelve strains of filamentous, heterocystous, nitrogen‐fixing cyanobacteria have been determined. When grown under diazotrophic conditions, protein, carbohydrate, lipid, and nucleic acids comprised 37–52%, 16–38%, 8–13%, and 8–11% of the dry weight, respectively. The presence of a combined nitrogen source resulted in an increase in the protein content of the cells and a decrease in the levels of lipids and carbohydrates, although biomass productivity was not affected significantly. Biochemical composition also changed during culture growth, with the highest levels of proteins and lipids occurring as the culture entered stationary phase, whereas the highest levels of carbohydrate and nucleic acids were found during the exponential phase. Total fatty acid levels in the strains assayed ranged between 3 and 5.7% of the dry weight. With regard to fatty acid composition, all strains showed high levels of polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SAFAs), with values of 24–45% and 31–52% of total fatty acids, respectively, whereas the levels of monounsaturated fatty acids (MUFAs) were in general lower (11– 32%). Palmitic acid (16:0) was the most prevalent SAFA, whereas palmitoleic (16:1n‐ 7) and oleic acid (18:1n‐9) were the most abundant MUFAs in all the strains. Among PUFAs, γ‐linolenic acid (GLA, 18:3n‐6) was present at high levels (18% of total fatty acids) in Nostoc sp. (Chile) and at lower levels (3.6% of total fatty acids) in Anabaenopsis sp. The presence of GLA has not been previously reported in these genera of cyanobacteria. The rest of the strains exhibited high levels (12–35% of total fatty acids) of α‐linolenic acid (ALA, 18:3n‐3). Linoleic acid (18:2n‐6) was also present at a substantial level in most of the strains. Eicosapentaenoic acid (EPA, 20:5n‐3) was also detected in Nostoc sp. (Albufera). Some filamentous nitrogen‐fixing cyanobacteria therefore represent potential sources of commercially interesting fatty acids.

Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta) by Improvement of Culture Conditions and Random Mutagenesis

Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

Overexpression of an exogenous phytoene synthase gene in the unicellular alga Chlamydomonas reinhardtii leads to an increase in the content of carotenoids.

Phytoene synthase (PSY) catalyses the first step in the production of carotenoids, which has been described as a key regulatory step in the carotenoids biosynthetic pathway. PSY gene from Dunaliella salina was constitutively expressed in Chlamydomonas reinhardtii under the control of the RBCS2 and HSP70A promoters and targeted to the chloroplast by the RBCS2 transit peptide. DsPSY overexpression resulted in a stable increase in the corresponding PSY transcript level and in the content of carotenoids such as violaxanthin, lutein, and β‐carotene, reaching between 125 and 260% the levels in control untransformed cells.

Properties of the in vivo Nitrate Reductase Assay in Maize, Soybean, and Spinach Leaves

Summary The effect of different parameters on the in vivo nitrate reductase activity in leaves of maize, soybean, and spinach has been examined. Leaf sections infiltrated under vacuum with an assay medium containing CO 2 tension and NO - 3 and incubated under air, excreted CO 2 tension and NO - 2 in the dark but not in the light. Nevertheless, addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, a potent inhibitor of the photosynthetic electron transport, promoted an immediate excretion of CO 2 tension and NO - 2 in the light. Under those conditions, subsequent addition of ascorbate-reduced dichlorophenolindophenol or N,N,N′,N′-tetramethylphenylenediamine, which can supply electrons to photosystem I, abolished the excretion of CO 2 tension and NO - 2 by the leaf sections. These results support the view that, in green tissues, the reducing equivalents photogenerated in the chloroplasts are more directly used for NO - 2 reduction than for NO - 2 reduction. Incubation of leaf discs under O 2 -atmosphere severely impaired NO - 2 accumulation. Under strict anaerobic conditions, the rate of NO - 2 production was only slightly enhanced as compared with that observed in the assay under air. Exogenous NO - 3 stimulated the activity, more markedly in soybean than in the other two species. Concentrations of exogenous NO - 3 between 50 and 100 mM were required for maximal activities in all the three species. The external pH optimum for in vivo NO - 3 reduction was 7.5 for maize and spinach and 7.0 for soybean. Inclusion of 1% (v/v) n -propanol in the infiltration-incubation medium enhanced the activity about 15%. However, 7% (v/v) n -propanol caused a drastic decrease in the rate of NO - 2 accumulation which could be greatly overcome by inclusion in the medium of NADH or reduced flavin mononucleotide.

PHOTOINACTIVATION OF SPINACH NITRATE REDUCTASE SENSITIZED BY FLAVIN MONONUCLEOTIDE. EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN

Abstract All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase plus catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.

ISOLATION AND CHARACTERIZATION OF A LYCOPENE β‐CYCLASE GENE FROM THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

The isolation, characterization, and regulation by light and nitrogen of the lycopene β‐cyclase gene from Chlorella zofingiensis Dönz (CzlcyB), involved in the biosynthesis of astaxanthin and lutein, have been performed in this work. These carotenoids are of high commercial value as dyes in food and as nutraceuticals. The open reading frame (ORF) of CzlcyB encoded a polypeptide of 546 amino acids. A single copy of CzlcyB has been found in C. zofingiensis. The chararacteristic Rossmann or dinucleotide binding fold, present in most lycopene cyclases, has been also identified in the LCYb of C. zofingiensis (CzLCYb). Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to cycle both lycopene and δ‐carotene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the lycopene β‐cyclases (LCYb) of the chlorophycean microalgae studied, being very closely related to LCYb of plants. Transcript levels of CzlcyB were increased under nitrogen deprivation, but no increase was observed under high‐light conditions. However, high irradiance triggered astaxanthin synthesis, while nitrogen deprivation by itself could not induce it. The combination of high irradiance and nitrogen deprivation led to a significant enhancement of the astaxathin accumulation.

Seasonal Differences in Photochemical Efficiency and Chlorophyll and Carotenoid Contents in Six Mediterranean Shrub Species under Field Conditions

The effects of summer and winter stress on the chlorophyll and carotenoid contents and photosystem 2 efficiency were examined in six Mediterranean scrub species. These six species belong to two different plant functional types: drought semi-deciduous (Halimium halimifolium L., Rosmarinus officinalis L., Erica scoparia L.) and evergreen sclerophylls (Juniperus phoenicea L., Pistacia lentiscus L., Myrtus communis L.). Two sites with different water availability were chosen. In the xerophytic site, despite they belong to two different functional types, R. officinalis and J. phoenicea showed a similar response. These were the most affected species in summer. H. halimifolium showed optimal values of Fv/Fm and non-significant seasonal changes in xanthophyll content. In the mesic site, E. scoparia and M. communis were apparently the most affected species by winter climatic conditions. P. lentiscus presented a pattern similar to H. halimifolium, except for elevated F0 values. In all the studied species, lutein plus zeaxanthin content was negatively correlated with Fv/Fm in summer and with leaf water potential, thus indicating that the thermal dissipation of energy was a general pattern for all species. Under stress, plant response is more species-specific than dependent on its functional type.

Isolation and Characterization of a Lycopene ε-Cyclase Gene of Chlorella (Chromochloris) zofingiensis. Regulation of the Carotenogenic Pathway by Nitrogen and Light

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Inactivation by acetylene of spinach nitrate reductase

Abstract The molybdoprotein NADH-nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1) from spinach can be inactivated by acetylene only when the enzyme is in its reduced state. Other gases such as ethylene, carbon monoxide, dinitrogen and others did not alter the enzyme activity. From the two partial activities of nitrate reductase, only the terminal nitrate reductase was impaired by acetylene while the dehydrogenase activity was rather stimulated. Functional dehydrogenase activity was required for inactivation when NADH was the reductant. Dithionite, dithionite + MV or dithionite + FMN were also able to sustain acetylene inactivation, whether or not nitrate reductase was previously depleted of its dehydrogenase activity. However, ascorbate or ascorbate + DCIP did not cooperate with acetylene for inactivating nitrate reductase. Nitrate and the competitive inhibitors with respect to nitrate of nitrate reductase, namely azide, cyanate and carbamyl phosphate, protected nitrate reductase from acetylene inactivation. Cyanide-inactivated nitrate reductase was still sensitive to acetylene, since, once the cyanide-inactivated enzyme was placed under acetylene, no ferricyanide reactivation could be attained. These results suggest that reduced nitrate reductase might bind acetylene at the nitrate active site, where molybdenum is supposed to be implicated, thus impairing the reduction of nitrate.

Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii

The carotenoid biosynthesis pathway catalyses the synthesis of essential pigments that are crucial for light harvesting and photoprotection in photosynthetic organisms. It allows the production of several commercially important compounds and is the target of many herbicides. In the present work we studied the influence of light on the carotenoid composition and the expression of genes encoding the main steps of the pathway in the freshwater microalga Chlamydomonas reinhardtii. We observed that there is an activation of the xanthophyll cycle in response to high light, but also in response to other stress conditions, such as nitrogen starvation, which has not been reported previously. We analysed the expression level of (1) genes encoding the two first enzymes of the pathway, phytoene synthase and phytoene desaturase; (2) the enzymes responsible for the cyclization of lycopene, lycopene β-cyclase and lycopene ε-cyclase; (3) zeaxanthin epoxidase, which catalyses the epoxidation of zeaxanthin; and (4) the three known carotene hydroxylases, directly involved in the synthesis of xanthophylls from α and β-carotene. Measurements of carotenoid content in the presence of inhibitors of protein and carotenoid synthesis suggest that only one of the two possible routes for the synthesis of zeaxanthin upon transference to high light, either the de novo synthesis of carotenoids or the interconversion of violaxanthin and zeaxanthin, is dependent on protein synthesis. The high increase in the transcript levels of the cytochrome-dependent carotene β- and ε-hydroxylases in response to high light suggests an important role of these enzymes in regulation of xanthophyll synthesis upon light stress. These conclusions may be of high interest if efficient engineering of the pathway is to be accomplished.