Origin of β-Carotene-Rich Plastoglobuli in Dunaliella bardawil

The halotolerant microalgae Dunaliella bardawil accumulates under nitrogen deprivation two types of lipid droplets: plastoglobuli rich in β-carotene (βC-plastoglobuli) and cytoplasmatic lipid droplets (CLDs). We describe the isolation, composition, and origin of these lipid droplets. Plastoglobuli contain β-carotene, phytoene, and galactolipids missing in CLDs. The two preparations contain different lipid-associated proteins: major lipid droplet protein in CLD and the Prorich carotene globule protein in βC-plastoglobuli. The compositions of triglyceride (TAG) molecular species, total fatty acids, and sn-1+3 and sn-2 positions in the two lipid pools are similar, except for a small increase in palmitic acid in plastoglobuli, suggesting a common origin. The formation of CLD TAG precedes that of βC-plastoglobuli, reaching a maximum after 48 h of nitrogen deprivation and then decreasing. Palmitic acid incorporation kinetics indicated that, at early stages of nitrogen deprivation, CLD TAG is synthesized mostly from newly formed fatty acids, whereas in βC-plastoglobuli, a large part of TAG is produced from fatty acids of preformed membrane lipids. Electron microscopic analyses revealed that CLDs adhere to chloroplast envelope membranes concomitant with appearance of small βC-plastoglobuli within the chloroplast. Based on these results, we propose that CLDs in D. bardawil are produced in the endoplasmatic reticulum, whereas βC-plastoglobuli are made, in part, from hydrolysis of chloroplast membrane lipids and in part, by a continual transfer of TAG or fatty acids derived from CLD.Eukaryotic cells accumulate neutral lipids in different tissues mainly in the form of lipid droplets (Murphy, 2012). Most lipid droplets consist of a core of triglycerides (TAGs) and/or sterol esters coated by a phospholipids monolayer and embedded with proteins (Zweytick et al., 2000). Plants accumulate TAGs in different tissues, primarily in seeds but also in fruit, such as palm oil, flowers, and leaves. The best characterized system for TAG metabolism is oil seeds, in which TAG serves as the major carbon and energy reservoir to be used during germination (Huang, 1992, 1996). Recent studies show that lipid droplets are not just static pools of lipids but have diverse metabolic functions (Farese and Walther, 2009). In addition, plants also contain plastoglobuli, small chloroplastic lipid droplets consisting primarily of storage lipids and pigments. Proteome analyses of plastoglobuli suggest that they are involved in synthesis and degradation of lipids, pigments, and coenzymes (Ytterberg et al., 2006; Lundquist et al., 2012). It has been shown that plant plastoglobuli are associated with thylakoid membranes (Austin et al., 2006; Ytterberg et al., 2006).It is not entirely clear where the TAGs are synthesized in the plant cell. Until recently, it has been assumed that most TAGs are made in the endoplasmatic reticulum (ER) from fatty acids, which are mostly synthesized in the chloroplast and imported to the cytoplasm (Joyard et al., 2010). However, the recent identification of the enzyme diacylglycerol acyl transferase in plant plastoglobuli (Lundquist et al., 2012) suggests that TAG may be synthesized directly in chloroplasts, although direct evidence is missing. TAG may be synthesized also from galactolipid fatty acids during stress or senescence by phytyl ester synthases, which catalyze acyl transesterification from galactolipids to TAGs (Lippold et al., 2012). Phosphatidyl choline (PC) plays a major role in acyl transfer of newly synthesized fatty acids from the chloroplast into TAGs at the ER in plants (Bates et al., 2009). An indication for the origin of glycerolipids in plants is the identity of the fatty acids at the sn-2 position: if it originates in the chloroplast, it is mostly C16:0, whereas if it was made in the ER, it is mostly C:18 (Heinz and Roughan, 1983).Many species of unicellular microalgae can accumulate large amounts of TAGs under growth-limiting conditions, such as nitrogen deprivation (Shifrin and Chisholm, 1981; Roessler, 1990; Avron and Ben-Amotz, 1992; Thompson, 1996). In green microalgae (Chlorophyceae), TAGs are usually synthesized and accumulated in cytoplasmatic lipid droplets (CLDs; Murphy, 2012), although in some cases, such as in Chlamydomonas reinhardtii starchless mutants, they also accumulate in chloroplasts (Fan et al., 2011; Goodson et al., 2011). Recent studies indicate that the CLDs are closely associated with ER membranes and possibly, chloroplast envelope membranes as well (Goodson et al., 2011; Peled et al., 2012).Green microalgae also contain two distinct types of chloroplastic lipid droplets. The first type is plastoglobuli, similar in morphology to higher plants plastoglobuli (Bréhélin et al., 2007; Kessler and Vidi, 2007). The second type is the eyespot (stigma), part of the visual system in microalgae. The eyespot is composed of a cluster of β-carotene-containing lipid droplets organized in several layers between grana membranes in the chloroplast (Häder and Lebert, 2009; Kreimer, 2009). Recent proteomic analysis of algal eyespot proteins revealed that they contain diverse structural proteins, lipid and carotenoid metabolizing enzymes, transporters, and signal transduction components (Schmidt et al., 2006).The origin of TAG in microalgae is still not clear. In C. reinhardtii, it was found that the major fatty acids in the sn-2 position are 16:0, which according to the plant dogma, is made in the chloroplast (Fan et al., 2011). In C. reinhardtii, which lacks PC, monogalactosyldiacylglycerol (MGDG) was proposed to replace PC in the mobilization of fatty acids from plastidal galactoglycerolipids into TAG based on mutation of a galactoglycerolipid lipase (Li et al., 2012). Based on these results and others, it has been proposed that, in C. reinhardtii, triglycerides are primarily produced in the chloroplast or combined with ER (Li et al., 2012; Liu and Benning, 2013).Plants and algae lipid droplets contain structural major proteins localized at the lipid droplet periphery, and their major function seems to be stabilization and prevention of fusion (Huang, 1992, 1996; Katz et al., 1995; Frandsen et al., 2001; Liu et al., 2009). In plant seed oils, the major classes of lipid droplet proteins are oleosins and caleosins, which have a characteristic hydrophobic loop with a conserved three Pro domain (Hsieh and Huang, 2004; Capuano et al., 2007; Purkrtova et al., 2008; Tzen, 2012). Oleosin and caleosin analogs were also recently identified in some green microalgal species (Lin et al., 2012; Vieler et al., 2012; Huang et al., 2013). However, the most abundant lipid droplets proteins in green algae (Chloropyceae) are a new family of major lipid droplet proteins (MLDPs) structurally distinct from plant oleosins and caleosins (Moellering and Benning, 2010; Peled et al., 2011; Davidi et al., 2012). Plastoglobules have different major lipid-associated proteins termed plastoglobules-associated protein-fibrillins, which form a distinct protein family with no sequence or structural similarities to oleosins (Kim and Huang, 2003). We have previously identified in the plastoglobuli rich in β-carotene (βC-plastoglobuli) a lipid-associated protein termed carotene globule protein (CGP), whose degradation destabilized the lipid droplets (Katz et al., 1995). The proteome of C. reinhardtii lipid droplet indicates that algal CLDs also contain several enzymes, suggesting that they are involved in lipid metabolism (Nguyen et al., 2011).The halotolerant green algae Dunaliella bardawil and Dunaliella salina ‘Teodoresco’ are unique in that they accumulate under high light stress or nitrogen deprivation large amounts of plastidic lipid droplets (βC-plastoglobuli), which consist of TAG and two isomers of β-carotene, all trans and 9-cis (Ben-Amotz et al., 1982, 1988). D. bardawil also accumulates CLD under the same stress conditions, similar to other green algae (Davidi et al., 2012). It has been shown that the function of βC-plastoglobuli is to protect the photosynthetic system against photoinhibition (Ben-Amotz et al., 1989). The enzymatic pathway for β-carotene synthesis in D. bardawil and D. salina has been partly identified, but the subcellular localization of β-carotene biosynthesis is not known (Jin and Polle, 2009). The synthesis of β-carotene depends on TAG biosynthesis (Rabbani et al., 1998); however, the origin of βC-plastoglobuli is not known. Are they formed within the chloroplast, or are they made in the cytoplasm? Is the TAG in βC-plastoglobuli and CLD identical or different, and where is it formed?D. bardawil is an excellent model organism for isolation of lipid droplet for several reasons. First, D. bardawil contains large amounts of both CLD and βC-plastoglobuli (Ben-Amotz et al., 1982; Fried et al., 1982), making it possible to obtain sufficient amounts of proteins and lipids from the two types of lipid pools for detailed analyses. Second, Dunaliella do not have a rigid cell wall and can be lysed by a gentle osmotic shock, which does not rupture the chloroplast. Therefore, it is possible to sequentially release pure CLD and βC-plastoglobuli by a two-step lysis (Katz et al., 1995). Third, D. bardawil seems to lack the eyespot structure, which can be clearly observed in other Dunaliella spp. even in a light microscope or by electron microscopy, but has never been observed in D. bardawil by us. It avoids the risk of cross contamination of βC-plastoglobuli with eyespot proteins. Fourth, the availability of protein markers for the major lipid droplet-associated proteins, CGPs and MLDPs, enabled both good immunolocalization and careful monitoring of the purity of the preparations by western analysis.In this work, we describe the purification, lipid compositions, and protein profiles of two lipid pools from D. bardawil: CLD and plastidic βC-plastoglobuli. A detailed proteomic analysis of these lipid droplets will be described in another work. Combined with detailed electron microscopy studies, these results led to surprising conclusions regarding the origin of the plastidic βC-plastoglobuli.     

Exploitation of Dunaliella for β-carotene production

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.     

Chemical induction of β-carotene biosynthesis

Marsh white seedless grapefruit were treated with the 2-diethylaminoethanol esters of the following acids: benzoic, phenylacetic, hydrocinnamic, 4-phenylbutyric, 5-phenylvaleric, valeric, hexanoic, heptanoic, octanoic, nonanoic, 5-chlorovaleric, cyclohexanecarboxylic, phenoxyacetic, p-chlorophenoxyacetic, 3-phenoxypropionic, cinnamic and p-chlorocinnamic. Several of these esters, in particular the hexanoate, 4-phenylbutyrate and cinnamate, caused the accumulation of large amounts of β-carotene. The effects of the hexanoate and of 2-phenoxytriethylamine, which causes only lycopene accumulation, were studied as functions of time. The hexanoate caused the rapid accumulation of lycopene during the first day. The amount of lycopene then began to decrease and that of β-carotene increased until, after 14 days, β-carotene was the major pigment. 2-Phenoxytriethylamine caused rapid lycopene accumulation during the first day and a slow steady increase afterwards. Thus, the mode of action of the β-carotene inducers may be similar to that of the lycopene inducers except that the former are probably rapidly hydrolysed by the esterase(s) in the flavedo, so that they no longer inhibit the cyclase(s), and β-carotene is accumulated at the expanse of lycopene.     

Proteome Analysis of Cytoplasmatic and Plastidic β-Carotene Lipid Droplets in Dunaliella bardawil

The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and β-carotene-rich (βC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in βC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The βC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, β-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of βC-plastoglobuli and the biosynthesis of β-carotene within βC-plastoglobuli and hypothesize that βC-plastoglobuli evolved from eyespot lipid droplets.Lipid droplets are the least characterized organelles in both mammalian and plant cells, and they were considered until a few years ago as passive storage compartments for triglycerides (TAG), sterol esters, and some pigments. However, recent studies have shown that they have diverse metabolic functions (Goodman, 2008; Farese and Walther, 2009; Murphy, 2012). Proteomic analyses in plants and some microalgae have shown that lipid droplets in the cytoplasm and in the chloroplast contain a large diversity of proteins including both structural proteins and many enzymes, indicating that they take an active metabolic role in the synthesis, degradation, and mobilization of glycerolipids, sterols, and pigments as well as in regulatory functions that have not yet been clarified (Schmidt et al., 2006; Ytterberg et al., 2006; Nguyen et al., 2011; Lundquist et al., 2012b; Eugeni Piller et al., 2014). A major limitation for determining the proteomes of lipid droplets, particularly in microalgae, is the purity and the homogeneity of the preparation. Green microalgae, for example, may contain three distinct pools of lipid droplets in one cell: the cytoplasmatic lipid droplets (CLD), the major neutral lipid pool, which are induced under stress conditions such as nitrogen limitation or at the stationary growth phase (Wang et al., 2009); plastoglobules, which are smaller lipid droplets within the chloroplast that have been shown to change in size and number under stress conditions and seem to be involved in stress resistance, metabolite transport, and the regulation of photosynthetic electron transport (Bréhélin et al., 2007; Besagni and Kessler, 2013); and the eyespot structure, part of the visual system in green algae, composed of one or several layers of lipid droplets, characterized by their orange color resulting from a high content of β-carotene (Kreimer, 2009). Disruption of microalgal cells, which is required for the isolation of the lipid droplets, usually involves harsh treatments such as sonication, mixing with glass beads, or use of a French press that breaks not only the cell membrane but also the chloroplast. Therefore, it is almost impossible to separate the different lipid droplet classes by the subsequent density gradient centrifugation, making it difficult to assign the origin of identified proteins. The other major difficulty is contamination by proteins released during cell lysis and fractionation, which associate and copurify with lipid droplets. These include cytoplasmic, chloroplastic, and mitochondrial proteins (Moellering and Benning, 2010; James et al., 2011; Nguyen et al., 2011; Nojima et al., 2013). Purification of isolated lipid droplets from loosely associated proteins is possible by treatments with detergents, high salt, and chaotropic agents (Jolivet et al., 2004; Nguyen et al., 2011); however, the danger in such treatments is that they also remove native loosely associated proteins from the lipid droplets.In this work, we tried to circumvent these problems by choosing a special algal species that is suitable for controlled cell lysis and fractionation and by utilizing two different contamination filters.The alga we selected, Dunaliella bardawil, is unique in that it accumulates large amounts of two different types of lipid droplets, CLD and β-carotene-rich (βC) plastoglobuli, under stress conditions (Davidi et al., 2014). The lack of a rigid cell wall in this alga allows lysis of the plasma membrane by a gentle osmotic shock, releasing CLD but leaving the chloroplast intact (Katz et al., 1995). This enables the recovery of large quantities of the two types of highly purified lipid droplets by differential lysis. In a recent study, we described the isolation and lipid compositions of these two lipid pools and showed that they have similar TAG compositions but different lipid-associated major proteins (Davidi et al., 2014).The high nutritional and pharmacological value of β-carotene for humans has promoted intensive research aimed to clarify its biosynthesis and regulation in plants and also led to attempts to increase β-carotene levels by genetic manipulations in crop plants such as tomato (Solanum lycopersicum; Rosati et al., 2000; Giorio et al., 2007) or by the creation of Golden rice (Oryza sativa; Ye et al., 2000). However, the capacity of plants to store β-carotene is limited, and in this respect, D. bardawil is an exceptional example of an organism that can accumulate large amounts of this pigment, up to 10% of its dry weight. This is enabled by the compartmentation and storage of this lipophilic pigment in specialized plastoglobules. Also, the unusual isomeric composition, consisting of around 50% 9-cis- and 50% all-trans-isomers (Ben-Amotz et al., 1982, 1988), is probably of major importance in this respect, due to the better solubility of the cis-isomer in lipids, which enables the storage of high concentrations exceeding 50% of the lipid droplets. The localization of carotenoid biosynthesis in plants appears to be tissue specific: in green tissues, it takes place in chloroplast membranes, probably within the inner chloroplast envelope membrane (Joyard et al., 2009), whereas in carotenoid-accumulating fruits, such as tomato or bell pepper (Capsicum annuum), it takes place in specialized organelles derived from chromoplasts (Siddique et al., 2006; Barsan et al., 2010). In green microalgae, there are at least two types of carotenoid-accumulating organelles: CLD and eyespot. Algae such as Haematococcus pluvialis and Chlorella zofigiensis accumulate carotenoids within CLD. In H. pluvialis, the major pigment, astaxanthin, is synthesized initially in the chloroplast as β-carotene and then transferred to CLD, where it is oxidized and hydroxylated to astaxanthin (Grünewald et al., 2001). The eyespot, which is composed of one or several layers of small β-carotene-containing lipid droplets, has been shown by proteomic analysis to include part of the β-carotene biosynthesis enzymes, indicating that β-carotene is probably synthesized within these lipid droplets (Schmidt et al., 2006). Similarly, plant chromoplasts also contain carotenoid biosynthesis enzymes (Schmidt et al., 2006; Ytterberg et al., 2006; Schapire et al., 2009). D. bardawil and Dunaliella salina are unique in that they accumulate large amounts of β-carotene within βC-plastoglobuli. A special focus in this work was the identification of the β-carotene biosynthesis machinery in D. bardawil. It is not known if the synthesis takes place inside the lipid βC-plastoglobuli or in chloroplast envelope membranes. Since D. bardawil also contains β-carotene and xanthophylls at the photosynthetic system, it is interesting to know whether the β-carotene that accumulates under stress in βC-plastoglobuli is produced by the constitutive carotenoid biosynthetic pathway or by a different stress-induced enzymatic system.     

Are allozymes differing in substrate specificity involved in the evolution of Silene species?

Summary The concerted action of two flavone-skeleton modifying genes, P and Me, and the alleles of three independently segregating loci g, gl and fg involved in flavone-glycosylation lead to the 33 different flavones so far identified in Silene. The alleles of the different loci involved in flavone-glycosylation control enzymes which differ in substrate specificity, a phenomenon not often described in higher organisms. The alleles of the different loci are variously distributed over the different species. The possible evolutionary implications of these distributions are discussed.     

Generation of active oxygen species (AOS) and induction of β-Glucanase activity by fungal elicitor xylanase in the suspension cultured cells of Tobacco

It was investigated that active oxygen species (AOS) involved in the plant defense responses induced by fungal elicitor xylanase. When xylanase from the fungusTrichoderma viridae was treated to tobacco suspension cultured cells as an elicitor, β-glucanase activity was increased markedly. Lignin biosynthesis was also increased and peaked at 72 h after the treatment with xylanase. The treatment of H₂O₂ also dramatically increased β-glucanase activity at 24 h, which was much earlier than that of xylanase did. Using lucigenin-and luminol-dependent chemiluminescence, the effects of xylanase on oxidative burst were examined. Superoxide anion (O₂) production was peaked at 40 h and 52 h after xylanase treatment and hydrogen peroxide (H₂O₂) release was peaked at 44 h and 56 h, suggesting H₂O₂ burst was followed by O₂ generation. The scavengers of AOS, n-propyl gallate (PG) and mannitol, inhibited xylanase-induced β-glucanase activity by 85% and 50%, respectively. The activity of superoxide dismutase (SOD), which catalyzes the dismutation of O₂ to H₂O₂, began to increase from 24 h and reached to maximum at 48 h after xylanase treatment. Pretreatment of N,N,-diethyldithiocarbamate (DDC), known as a SOD inhibitor, caused the inhibition of H₂O₂ generation by 80% and reduced the β-glucanase activity by 60%. Treatment of 2,5-norbonadiene (NBD), a specific ethylene-action inhibitor, did not have any significant effect on xylanase-induced β-glucanase activity. This result suggested that ethylene did not involve in xylanase-induced response. Our results strongly suggest that the AOS generation is an essential component in plant defense response, in which cell wall degrading enzyme, glucanase, contributes to remove the necrotic tissue induced by pathogens.     

Are polyamines involved in the induction and regulation of the Crassulacean acid metabolism?

Leaves of plants with Crassulacean acid metabolism (CAM) were analyzed for variation in the content of polyamines in connection with the metabolism of malic acid in the dark and in the light, and with the induction of full-CAM activity. Under conditions (long days) resulting in extremely low CAM activity, young leaves of K. blossfeldiana have very low content in the polyamine-precursor arginine and in putrescine. The content in these two substances was increased dramatically by full-CAM induction with short days. During the course of the night/day cycle two peaks of putrescine content were observed in leaves of Kalanchoe blossfeldiana Poelln. Tom Thumb performing full-CAM operation: a large increase occurs toward the end of the day and the first half of the night, and its kinetics corresponds to the increase in the rate of malic acid synthesis; another peak, very sharp, appears during the first hours of the day, concomitant with the time of release of malic acid from the vacuole into the cytoplasm. In the case of Bryophyllum daigremontianum Berger similar variations were observed for the content in spermidine. These results support the hypothesis that polyamines could be involved in countering the tendency toward acidification of the cytoplasm at those moments of CAM operation at which the local concentration of malic acid is increased (i.e., during active synthesis in the dark and during the efflux from the vacuole in the light).Abbreviation CAM Crassulacean acid metabolism     

Are mitochondria a permanent source of reactive oxygen species?

The observation that in isolated mitochondria electrons may leak out of the respiratory chain to form superoxide radicals (O(2)(radical-)) has prompted the assumption that O(2)(radical-) formation is a compulsory by-product of respiration. Since mitochondrial O(2)(radical-) formation under homeostatic conditions could not be demonstrated in situ so far, conclusions drawn from isolated mitochondria must be considered with precaution. The present study reveals a link between electron deviation from the respiratory chain to oxygen and the coupling state in the presence of antimycin A. Another important factor is the analytical system applied for the detection of activated oxygen species. Due to the presence of superoxide dismutase in mitochondria, O(2)(radical-) release cannot be realistically determined in intact mitochondria. We therefore followed the release of the stable dismutation product H(2)O(2) by comparing most frequently used H(2)O(2) detection methods. The possible interaction of the detection systems with the respiratory chain was avoided by a recently developed method, which was compared with conventional methods. Irrespective of the methods applied, the substrates used for respiration and the state of respiration established, intact mitochondria could not be made to release H(2)O(2) from dismutating O(2)(radical-). Although regular mitochondrial respiration is unlikely to supply single electrons for O(2)(radical-) formation our study does not exclude the possibility of the respiratory chain becoming a radical source under certain conditions.     

Influence of areal density on β-carotene production by Dunaliella salina

High irradiance is probably the most important factor responsible for the massive accumulation of β-carotene by the halotolerant green alga Dunaliella salina. Operating outdoor cultures at optimal areal densities should result in maximal productivity. It is known that the optimal areal density is not fixed for all algae, where it could vary depending on the type of algae cultured, pond construction, turbulence and prevailing environmental conditions. At biomass concentrations below the optimum, more light per cell is available than that which could be absorbed by the biomass. These high light conditions should favour carotenogenesis and could result in higher β-carotene production rates. The results obtained clearly showed that over and above light and nutrient stress, an extremely important aspect is the residence time of the cells in the ponds. Longer residence times resulted in the development of larger cells, containing larger quantities of β-carotene. Productivity of biomass and β-carotene were about 70% higher at areal densities of 35–45 g m^-2, compared to areal densities of 15–25 g m^-2.     

Spray-drying of Dunaliella salina to produce a β-carotene rich powder

Powders of Dunaliella salina biomass were obtained by spray drying a cell concentrate under different drying regimes. A three-factor, two-level experimental design was employed to investigate the influence of inlet temperature, outlet temperature and feed solids on β-carotene recovery. The effect of microencapsulation in a polymer matrix of maltodextrin and gum arabic was also studied. All powders were stored under specific conditions to assess the stability of the native β-carotene. There was a trend indicating that lower outlet temperature yielded higher carotenoid recoveries, β-carotene recovery varying between 57% and 91%. Microencapsulated biomass yielded 100% recoveries. All non-microencapsulated powders were unstable in terms of β-carotene content in the presence of natural light and oxygen showing 90% degradation over a 7-day period. The incorporation of a microencapsulating agent had a significant increase in the storage stability. Results indicated a first-order degradation of the β-carotene in microencapsulated powders with kinetic constants of 0.06 day⁻¹ and 0.10 day⁻¹. HPLC analysis showed no effect of drying processes on isomer composition (9-cis-β-carotene and all-trans-β-carotene ratio). This behaviour was also observed during storage of the microencapsulated powders. Received 16 October 1996/ Accepted in revised form 13 November 1997     

Are olfactory cues involved in nest recognition in two social species of estrildid finches?

Reliably recognizing their own nest provides parents with a necessary skill to invest time and resources efficiently in raising their offspring and thereby maximising their own reproductive success. Studies investigating nest recognition in adult birds have focused mainly on visual cues of the nest or the nest site and acoustic cues of the nestlings. To determine whether adult songbirds also use olfaction for nest recognition, we investigated the use of olfactory nest cues for two estrildid finch species, zebra finches (Taeniopygia guttata) and Bengalese finches (Lonchura striata var. domestica) during the nestling and fledgling phase of their offspring. We found similar behavioural responses to nest odours in both songbird species. Females preferred the odour of their own nest over a control and avoided the foreign conspecific nest scent over a control during the nestling phase of their offspring, but when given the own odour and the foreign conspecific odour simultaneously we did not find a preference for the own nest odour. Males of both species did not show any preferences at all. The behavioural reaction to any nest odour decreased after fledging of the offspring. Our results show that only females show a behavioural response to olfactory nest cues, indicating that the use of olfactory cues for nest recognition seems to be sex-specific and dependent on the developmental stage of the offspring. Although estrildid finches are known to use visual and acoustic cues for nest recognition, the similar behavioural pattern of both species indicates that at least females gain additional information by olfactory nest cues during the nestling phase of their offspring. Thus olfactory cues might be important in general, even in situations in which visual and acoustic cues are known to be sufficient.     

Strain selection for β-carotene production by Dunaliella

Four strains of Dunaliella were grown at 25°C and pH 8±0.5, with continous illumination at 200 W/m². Their maximum specific growth rates ranged from 0.093 day^-1 to 0.234 day^-1, nitrate yields from 3.0 to 7.8 g cells/g NaNO₃ and lipid contents from 3% to 6% of the dry wt, with carotenes 50 to 80% of the lipids. Of the carotenes, -carotene made up 7 to 19%; all-trans--carotene 32 to 52% and 9-cis--carotene 29 to 55%. There are, therefore, considerable intra-specific differences between strains of Dunaliella.     

New mode of Dunaliella biotechnology: two-phase growth for β-carotene production

Attempts to adapt the laboratory experience of bi-phasic growth and carotenogenesis in Dunaliella to large-scale conditions were highly successful. Algae were initially cultivated in stage one for optimizing biomass production of cells containing a low β-carotene to chlorophyll ratio. The culture was then transferred to stage two, diluted to about one third and induced for carotenogenesis. The bi-phase cultivation increased β- carotene productivity to 450 mg m^-2 d^-1 in stage one and to 300 mg m^-2 d^-1 in stage two, compared to the relatively low productivity of below 200 mg β- carotene m^-2 d^-1, in the conventional one phase type of cultivation. The results indicate that a selected algal product can be promoted specifically by growth manipulation of the alga and its environment.     

Cellular reactive oxygen species inhibit MPYS induction of IFNβ

Many inflammatory diseases, as well as infections, are accompanied by elevation in cellular levels of Reactive Oxygen Species (ROS). Here we report that MPYS, a.k.a. STING, which was recently shown to mediate activation of IFNβ expression during infection, is a ROS sensor. ROS induce intermolecular disulfide bonds formation in MPYS homodimer and inhibit MPYS IFNβ stimulatory activity. Cys-64, -148, -292, -309 and the potential C₈₈xxC₉₁ redox motif in MPYS are indispensable for IFNβ stimulation and IRF3 activation. Thus, our results identify a novel mechanism for ROS regulation of IFNβ stimulation.     

Pilot culture of three strains of Dunaliella salina for β-carotene production in open ponds in the central region of Iran

Summary Three strains of Dunaliella salina (I, G and A) were cultivated under the climatic conditions of Iran, in open ponds to compare the β-carotene production and the specific rate of growth. The experiments were accomplished in two separate stages. In the first stage, the cells were grown in ponds on nutrient-rich medium containing 2 M NaCl to obtain the necessary biomass. In the second stage, cells were stressed on nutrient-poor medium containing 2.5 M NaCl for β-carotene induction. The results showed that the specific growth rate of strain I was the highest during the first stage, whereas during the second stage, the growth rates of three strains were approximately the same. The overall results indicated that strain G had the highest potential for β-carotene accumulation of the strains tested and hence it was concluded that this strain is more suitable for outdoor cultivation under the climatic conditions of Iran than the other two.     

Are mitochondria a spontaneous and permanent source of reactive oxygen species?

The bioenergetic properties of mitochondria in combination with the high turnover rate of dioxygen qualify these organelles for the formation of reactive oxygen species (ROS). The assumption that mitochondria are the major intracellular source of ROS was essentially based on in vitro experiments with isolated mitochondria. The transfer of these data to the living cell may, however, be incorrect. Artefacts due to the preparation procedure or inadequate detection methods of ROS may lead to false positive results. Inhomogeneous results were found to be due to an interaction of the detection system with components of the respiratory chain which could be avoided by a recently developed non-invasive method. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation from ubiquinol to the cytochrome bc(1) complex. This electron bifurcation requires the free mobility of the head domain of the Rieske iron-sulfur protein. Inhibition of electron bifurcation by antimycin A causes leakage of single electrons to oxygen which results in the release of ROS. Hindrance of electron bifurcation was also observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc(1) complex is inserted. Irrespective of whether the fluidity of the membrane was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting the involvement of the ubiquinol/cytochrome bc(1) redox couple in this process.     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Photosynthetic characteristics of Dunaliella salina (Chlorophyceae,Dunaliellales) in relation to β-carotene content

Photosynthetic characteristics of Dunaliella salina with high (red form) and low β-carotene (green form) concentrations were studied. D. salina growing in brine saltworks exhibited a high level of β-carotene (15 pg cell⁻¹). The rate of oxygen evolution as a function of irradiance was higher in the red than in the green form (on chlorophyll basis). Photosynthetic inhibition of the green form was observed above 500 μmol m⁻² s⁻¹. The red form appeared more resistant to high irradiance and no inhibition in O₂ evolution was observed up 2000 μmol m⁻² s⁻¹. However, when these results are expressed on a cell number basis the rate of oxygen evolution was significantly higher in the green form. Carbonic anhydrase (CA) activity (total, soluble, membrane bound) was found in red and green forms. CA was higher in the red form on a chlorophyll basis, but lower if expressed on a protein basis. The light dependent rate of oxygen evolution and photoinhibition depends on the concentration of β-carotene in D. salina cells.     

Molecular identification of β-carotene hyper-producing strains of Dunaliella from saline environments using species-specific oligonucleotides

Sequence-specific-oligonucleotides analysis has been used to identify Dunaliella bardawil, D. salina and D. parva from hypersaline environments based on their structural features of introns from the 18S rDNA. Carotenogenic and halophilic strains such as D. bardawil and D. salina were identified as harboring II and I introns within 18S rDNA, respectively. This is the first report on the existence of D. bardawil in saline water bodies of Mexico and Latin America.