Enhancement of β-Carotene Synthesis by Citrus Products

β-Ionone, a stimulatory compound in the microbiological production of β-carotene by mated cultures of Blakeslea trispora, could be replaced with low-cost agricultural by-products (citrus oils, citrus pulp, or citrus molasses) with as good or better carotene yields. Peak yields (81 to 129 mg of carotene per g of dry solids) were achieved in 5 days. The various citrus products tested did not change the pigments produced; all trans-β-carotene remained the pre-dominant pigment. The acid-hydrolyzed soybean meal and corn used in previous production media could be replaced with unhydrolyzed cottonseed embryo meal and corn in a medium that also contained a natural lipid, deodorized kerosene, nonionic detergent, and a precursor.     

Characterization of the Role of β-Carotene 9,10-Dioxygenase in Macular Pigment Metabolism

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.     

Enhanced Production of β-Carotene by Recombinant Industrial Wine Yeast Using Grape Juice as Substrate

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their β-carotene production in real grape juice. The results showed that highest β-carotene content (5.89 mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous β-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.     

Characterization of β -Carotene Ketolases, CrtW, from Marine Bacteria by Complementation Analysis in Escherichia coli

A complementation analysis was performed in Escherichia coli to evaluate the efficiency of β-carotene ketolases (CrtW) from the marine bacteria Brevundimonas sp. SD212, Paracoccus sp. PC1 (Alcaligenes PC-1), and Paracoccus sp. N81106 (Agrobacterium aurantiacum), for astaxanthin production. Each crtW gene was expressed in Escherichia coli synthesizing zeaxanthin due to the presence of plasmid pACCAR25ΔcrtX. Carotenoids that accumulated in the resulting E. coli transformants were examined by chromatographic and spectroscopic analyses. The transformant carrying the Paracoccus sp. PC1 or N81106 crtW gene accumulated high levels of adonixanthin, which is the final astaxanthin precursor for CrtW, and astaxanthin, while the E. coli transformant with crtW from Brevundimonas sp. SD212 did not accumulate any adonixanthin and produced a high level of astaxanthin. These results show efficient conversion by CrtW of Brevundimonas sp. SD212 from adonixanthin to astaxanthin, which is a new-found characteristic of a bacterial CrtW enzyme. The phylogenetic positions between CrtW of the two genera, Brevundimonas and Paracoccus, are distant, although they fall into α-Proteobacteria.     

Involvement of β-Carotene in the Antioxidant Defense of the Bacterial Cell

Effects of recombinant -carotene on the resistance of E. coli culture to menadione and paraquat were studied. The presence of -carotene in E. coli cells prevented, to a considerable extent, an increase in superoxide dismutase activity (induced by redox mediators) without affecting the culture growth. These findings suggest that -carotene is involved in the defense of cells against oxidative stress.     

Characterization of the Main Light-Harvesting Chlorophyll a／b-Protein Complex of Green Alga, Bryopsis corticulans

The main light-harvesting chlorophyll a/b -protein complex (LHC Ⅱ) has been isolated directly from thylakoid membranes of shiphonous 8Teen alga, Bryopsis corticulans Setch. by using two consecutive runs of anion exchange and gel-filtration chromatography. Monomeric and trimeric subcomplexes of LHC Ⅱ were obtained by using sucrose gradient ultracentrifugation. Pigment analysis by reversed-phase high performance liquid chromatography showed that chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin, violaxanthin and siphonaxanthin were involved in LHC Ⅱ from B. corticulans. The properties of electronictransition of monomeric LHC Ⅱ showed similarities to those of trimeric LHC Ⅱ. Circular dichroism spectroscopy showed that strong intramolecular interaction of excitonic dipoles between Chl a and between Chl b exist in one LHC Ⅱ apoprotein, while the intermolecular interaction of these dipoles can be intensified in the trimeric structure. The monomer has high efficient energy transfer from Chl b and siphonaxanthin to Chl a similarly to that of the trimer. Our results suggest that in B. corticulans, LHC Ⅱ monomer has high ordered pigment organization that play effective physiological function as the trimer, and thus it might be also a functional organization existing in thylakoid membrane of B.corticulans.     

Characterization of the multiple forms of β-xylanase produced by a Streptomyces sp. growing on lignocellulose

Summary Streptomyces sp. strain EC10 degraded efficiently the hemicellulose fraction of wheat straw. Three forms of -xylanases detected in the culture filtrate were purified by precipipation with ammonium sulphate, chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-100. The three purified enzymes (X _ia , X _ib and X _ii ) were homogeneous by polyacrylamide gel electrophoresis. The enzymes were typical non-debranching endo--xylanases (1,4--d-xyla xylanohydrolases; E.C.3.2.1.8) with respective relative molecular weights of 32,000, 22,000 and 21,000 and isoelectric points of 6.8, 8.9 and 5.2. The enzymes were highly specific for xylans and showed optimal activity at pH 7.0–8.0 and 60°C. The preparations were completely free from cellulolytic activity (endoglucanase) and showed high thermal stability. No synergy between the three enzymes was detected for complete xylan hydrolysis of deacetylated arabino- and glucuronoxylans.Offprint requests:to: M. J. Penninckx     

Characterization of a β-glycosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius

In cell free extracts of the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius ATCC27009, we have identified β-gluco- and galactosidase activities showing a specific activity of 0.1 and 12 U/mg, respectively. The two enzymatic activities are associated with different polypeptides and we show here the functional cloning, the expression in Escherichia coli and the characterisation of the β-glucosidase (Aaβ-gly). The enzyme, which is optimally active and stable at temperatures above 65°C, belongs to glycoside hydrolase family 1 (GH1) and shows wide substrate specificity on different aryl-glycosides and cello-oligosaccharides with k _cat/K _M for 4-nitrophenyl-β-D-glucoside and cellobiose of 2,976 and 185 s⁻¹mM⁻¹, respectively. Interestingly, upstream to the β-glycosidase gene, we identified a second ORF homologous to the ATPase subunit of the bacterial ABC transporters (abc1) that is co-transcribed with the β-glycosidase gene glyB and that could be involved in the carbohydrate import. The activity of the enzyme on cello-oligosaccharides of up to five glucose units strongly indicates that the enzyme could be involved in vivo in the degradation of glucans together with endoglucanase enzymes previously described. This, together with the co-expression of the two genes, suggests a role for the glyB-abc1 cluster in A. acidocaldarius in the degradation of cellulose and hemicelluloses. Enzymes: EC 3.2.1.21; EC 3.2.1.23; EC 3.2.1.25; EC 3.2.1.38; EC 3.2.1.37 An erratum to this article can be found at     

A Circular Dichroism Spectroscopic Study Revealing the Cause of the Changes of Chlorophyll a Fluorescence Induction of Photosystem Ⅱ During Heat Treatment

Chlorophyll a fluorescence and circular dichroism (CD) spectra of photosystem Ⅱ (PSⅡ) membrane were measured after heat treatment. The chlorophyll fluorescence parameter Fo' remained stable after treatment at the temperatures from 30 ℃ to 40 ℃ and then reached a maximum after treatment at 55 ℃. In PSⅡ membranes and LHCⅡ (light-harvesting chlorophyll a/b binding complex)-enriched complexes, anomalous CD signals with extremely large amplitudes occurred during the heat treatment. The temperature corresponding to the maximum anomalous CD intensity peaking at 677 nm was 40 ℃. The results indicate that the aggregation state of the LHCⅡ in PSⅡ is related to the anomalous CD signal, and can be an important factor influencing Fo' in the heat treatment of PSⅡ membrane.     

Changes of Photosystem Ⅱ Electron Transport in the Chlorophyll-deficient Oilseed Rape Mutant Studied by Chlorophyll Fluorescence and Thermoluminescence

The photosystem Ⅱ (PSII) complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has de creased the amount of light-harvesting complexes with an increased amount of some core polypeptides of PSII,including CP43 and CP47. By means of chlorophyll fluorescence and thermoluminescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type.In the chlorophyll b-deficient mutant, the decay of the primary electron acceptor quinones (QA-) reoxidation was decreased, measured by the fluorescence. Furthermore, the thermolumlnescence studies in the chlorophyll b deficient mutant showed that the B band (S2/S3QB-) decreased slightly and shifted up towards higher temperatures.In the presence of dichlorophenyl-dimethylurea, which is inhibited in the electron flow to the second electron acceptor quinines (QB) at the PSII acceptor side, the maximum of the Q band (S2QA-) was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyll b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA- via forward electron flow and slower reduction of the oxidation S states.     

Fluorescence Quenching Studies of Trp Repressor–Operator Interaction

Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding. Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components. The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern–Volmer quenching constant equal to about 2.0–2.3 M^?1. The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern–Volmer quenching constant characteristic of L-tryptophan in the solvent. When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed. Acrylamide quenching experiments indicate that the Stern–Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M^?1. The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.     

Nano-coating of β-galactosidase onto the Surface of Lactose by Using an Ultrasound-assisted Technique

We nano-coated powdered lactose particles with the enzyme β-galactosidase using an ultrasound-assisted technique. Atomization of the enzyme solution did not change its activity. The amount of surface-attached β-galactosidase was measured through its enzymatic reaction product D-galactose using a standardized method. A near-linear increase was obtained in the thickness of the enzyme coat as the treatment proceeded. Interestingly, lactose, which is a substrate for β-galactosidase, did not undergo enzymatic degradation during processing and remained unchanged for at least 1 month. Stability of protein-coated lactose was due to the absence of water within the powder, as it was dry after the treatment procedure. In conclusion, we were able to attach the polypeptide to the core particles and determine precisely the coating efficiency of the surface-treated powder using a simple approach.     

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.     

Spectroscopic Characterization of the Excitation Energy Transfer in the Fucoxanthin–Chlorophyll Protein of Diatoms

We characterized the energy transfer pathways in the fucoxanthin–chlorophyll protein (FCP) complex of the diatom Cyclotella meneghiniana by conducting ultrafast transient absorption measurements. This light harvesting antenna has a distinct pigment composition and binds chlorophyll a (Chl-a), fucoxanthin and chlorophyll c (Chl-c) molecules in a 4:4:1 ratio. We find that upon excitation of fucoxanthin to its S₂ state, a significant amount of excitation energy is transferred rapidly to Chl-a. The ensuing dynamics illustrate the presence of a complex energy transfer network that also involves energy transfer from the unrelaxed or ‘hot’ intermediates. Chl-c to Chl-a energy transfer occurs on a timescale of a 100 fs. We observe no significant spectral evolution in the Chl-a region of the spectrum. We have applied global and target analysis to model the measured excited state dynamics and estimate the spectra of the states involved; the energy transfer network is discussed in relation to the pigment organization of the FCP complex.     

Structural Analysis of a Trimer of β2-Microgloblin Fragment by Molecular Dynamics Simulations

A peptide ${\beta }_2$-${\text{m}}_{21?31}$, which is a fragment from residue 21 to residue 31 of ${\beta }_2$-microgloblin, is experimentally known to self-assemble and form amyloid fibrils. In order to understand the mechanism of amyloid fibril formations, we applied the replica-exchange molecular dynamics method to the system consisting of three fragments of ${\beta }_2$-${\text{m}}_{21?31}$. From the analyses on the temperature dependence, we found that there is a clear phase transition temperature in which the peptides aggregate with each other. Moreover, we found by the free energy analyses that there are two major stable states: One of them is like amyloid fibrils and the other is amorphous aggregates.     

Characterization of a panel of six β2-adrenergic receptor antibodies by indirect immunofluorescence microscopy

Background

The β₂-adrenergic receptor (β₂AR) is a primary target for medications used to treat asthma. Due to the low abundance of β₂AR, very few studies have reported its localization in tissues. However, the intracellular location of β₂AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β₂AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β₂AR in primary cell cultures.

Methods

A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β₂AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β₂AR were identified and used to localize native β₂AR in primary cultures of rat airway smooth muscle and epithelial cells. β₂AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ^([3H]DHA).

Results

Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β₂AR (Ab-Bethyl) specifically recognized human but not rat β₂AR. An antibody developed against the C-terminal domain of the mouse β₂AR (Ab-sc570) specifically recognized rat but not human β₂AR. An antibody developed against 78 amino acids of the C-terminus of the human β₂AR (Ab-13989) was capable of recognizing both rat and human β₂ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β₂AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.

Conclusion

Antibodies have been identified that recognize human β₂AR, rat β₂AR or both rat and human β₂AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β₂AR in tissues.     

Antioxidant Activity of Supramolecular Water-Soluble Fullerenes Evaluated by β-Carotene Bleaching Assay

We investigated the antioxidant activity of supramolecular water-soluble fullerenes, polyvinylpyrrolidone (PVP)-entrapped C₆₀, and γ-cyclodextrin (CD)-bicapped C₆₀, based on comparable β-carotene bleaching assay. Antioxidant activity against reactive oxygen species (ROS) generated by three different methods, (i) autoxidation of linoleic acid, (ii) hydrogen peroxide promoter, and (iii) photoirradiation, was evaluated as percent of inhibition relative to a control experiment in view of the bleaching rate constant (k _obs) as well as the persistent absorbancy of β-carotene. Water-soluble fullerenes exhibit significant inhibitory effects on the oxidative discoloration of β-carotene in any system.     

Characterization of a novel β-glucosidase from a Stachybotrys strain

An improved mutant was isolated from the cellulolytic fungus Stachybotrys sp. after nitrous acid mutagenesis. It was fed-batch cultivated on cellulose and its extracellular cellulases (mainly the endoglucanases and β-glucosidases) were analyzed. One β-glucosidase was purified to homogeneity after two steps, MonoQ and gel filtration and shown to be a dimeric protein. The molecular weight of each monomer is 85 kDa. Besides its aryl β-glucosidase activity towards salicin, methyl-umbellypheryl-β-d-glucoside (MUG) and p-nitrophenyl-β-d-glucoside (pNPG), it showed a true β-glucosidase activity since it splits cellobiose into two glucose monomers. The V_max and the K_m kinetics parameters with pNPG as substrate were 78 U/mg and 0.27 mM, respectively. The enzyme shows more affinity to pNPG than cellobiose and salicin whose apparent values of K_m were, respectively, 2.22 and 37.14 mM. This enzyme exhibits its optimal activity at pH 5 and at 50 °C. Interestingly, this activity is not affected by denaturing gel conditions (SDS and β-mercaptoethanol) as long as it is not pre-heated. The N-terminal sequence of the purified enzyme showed a significant homology with the family 1 β-glucosidases of Trichoderma reesei and Humicola isolens even though these two enzymes are much smaller in size.     

Characterization of the β-Carotene Hydroxylase Gene DSM2 Conferring Drought and Oxidative Stress Resistance by Increasing Xanthophylls and Abscisic Acid Synthesis in Rice

Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.Abiotic stresses such as drought, salinity, and adverse temperatures are major limiting factors for plant growth and reproduction. To respond to environmental cues, plants have evolved a variety of biochemical and physiological mechanisms to adapt to adverse conditions during their growth and development (Boyer, 1982). Abscisic acid (ABA) has been recognized as a stress hormone that coordinates the complex networks of stress responses. Under drought or salt stress conditions, plant endogenous ABA level can rise to about 40-fold, triggering the closure of stomata and accumulating reactive oxygen species (ROS), dehydrins, and late embryogenesis abundant proteins for osmotic adjustment (Verslues et al., 2006). The endogenous ABA level is determined by ABA biosynthesis, catabolism, and release of ABA from ABA-Glc conjugates (Nambara and Marion-Poll, 2005; Lee et al., 2006). Therefore, identification of all the components affecting active ABA content is essential for a complete understanding of the action of the hormone.Numerous ABA biosynthetic genes have been identified through mutant analysis, such as maize (Zea mays) viviparous mutants vp2, vp5, vp7, vp9, vp14, w3, y3, and y9 (Schwartz et al., 1997; Hable et al., 1998; Singh et al., 2003); rice (Oryza sativa) preharvest-sprouting mutants psh1, psh2, psh3, and psh4 (Fang et al., 2008); sunflower (Helianthus annuus) nondormant mutant nd-1 (Conti et al., 2004); Arabidopsis (Arabidopsis thaliana) ABA- and nonphotochemical quenching (NPQ)-deficient mutants aba1, aba2, aba3, aba4, npq1, npq2, b1, b2, and nced3 (Havaux et al., 2000; Xiong et al., 2001; Tian et al., 2003; Barrero et al., 2005; Kim and DellaPenna, 2006; North et al., 2007); and tomato (Solanum lycopersicum) white-flower mutant wf (Galpaz et al., 2006; Supplemental Fig. S1). The mutants unable to biosynthesize carotenoid precursors for endogenous ABA synthesis often produced preharvest-sprouting seeds and wilted or white leaves (Gubler et al., 2005; Nambara and Marion-Poll, 2005; Finch-Savage and Leubner-Metzger, 2006).ABA biosynthesis initiates with the synthesis of a C5 building block, isopentenyl pyrophosphate, and its isomer dimethylallyl pyrophosphate through a plastid methylerythritol phosphate pathway (Eisenreich et al., 2001; Hunter, 2007). The three isopentenyl pyrophosphate molecules are then added to dimethylallyl pyrophosphate by geranylgeranyl diphosphate synthase to produce C20 geranylgeranyl diphosphate. Two geranylgeranyl diphosphates are condensed by a committing enzyme, phytoene synthase, to produce colorless C40 carotenoid phytoene, which is then desaturated and isomerized into red-colored lycopene by phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), and Z-ISO and CRTISO isomerases in plants (Isaacson et al., 2002; Park et al., 2002). Subsequently, several cyclization and hydroxylation reactions take place to yield α-carotene and β-carotene (Li et al., 1996; Hable et al., 1998; Park et al., 2002; Miki and Shimamoto, 2004; Fang et al., 2008). Heme-type cytochrome P450-type CYP97 and non-heme-type β-carotene hydroxylase (BCH) are primarily responsible for the hydroxylation of α-carotene and β-carotene to produce lutein and zeaxanthin, respectively. Zeaxanthin, an important component of the xanthophyll cycle, is epoxidated by zeaxanthin epoxidase to produce violaxanthin, and this reaction can be reversed by violaxanthin deepoxidase to increase the xanthophyll cycle for plants to adapt to high-light stress (Johnson et al., 2008). Neoxanthin synthase converts violaxanthin into neoxanthin (North et al., 2007). In chloroplast, 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves violaxanthin and neoxanthin to produce xanthoxin, the direct substrate for ABA synthesis via ABA aldehyde (Schwartz et al., 1997, 2003; Xiong and Zhu, 2003). Increasing evidence suggest that the endogenous ABA level is fine-tuned by differential regulation of the multiple steps of ABA biosynthesis (Seo and Koshiba, 2002; Nambara and Marion-Poll, 2005; Destefano-Beltrán et al., 2006; Thompson et al., 2007; Rodríguez-Gacio et al., 2009; Supplemental Fig. S1).The xanthophyll cycle (light-dependent reversible conversion between violaxanthin and zeaxanthin) is involved in photoprotection in PSII by regulating the nonradiative dissipation of excess absorbed light energy as heat (Gilmore et al., 1994). Mutants with defects in the xanthophyll cycle exhibit a weak photoprotective ability and produce ROS such as hydrogen peroxide (H₂O₂) when the absorption of light energy exceeds that consumed for photosynthesis (Niyogi, 1999). Under dehydration stress, electrons at a high energy state can easily form ROS, which are toxic to proteins, DNA, and lipids (Mittler, 2002; Apel and Hirt, 2004). However, plants have evolved a variety of biochemical and physiological mechanisms to scavenge ROS, thus maintaining a balance between ROS production and scavenging (Mittler et al., 2004).An association between the xanthophyll cycle and stress tolerance has been reported in plants. In Arabidopsis, overexpression of a bacterial BCH gene caused a specific 2-fold increase in the size of the xanthophyll cycle and enhanced photooxidative tolerance (Davison et al., 2002). Constitutive overexpression of a bacterial BCH gene, crtZ, in tobacco (Nicotiana tabacum) led to increased zeaxanthin synthesis and enhanced UV light tolerance (Götz et al., 2002). In Arabidopsis, zeaxanthin synthesis can be catalyzed by both heme-type CYP97 hydroxylases LUT1 and LUT5 and non-heme-type hydroxylases BCH1 and BCH2, and these two types exhibit some overlapping activities (Tian et al., 2003, 2004; Kim and DellaPenna, 2006). In contrast to the intensive molecular and genetic studies of BCH in Arabidopsis, the counterpart in economically important crops such as rice has not been identified.In this study, we characterized the rice drought-sensitive mutant dsm2, impaired in the gene DSM2 encoding a BCH. Our results demonstrate that DSM2 acts as a putative enzyme catalyzing the biosynthesis of zeaxanthin, one of the precursors of ABA that participates in the process of NPQ. Decreases of NPQ, maximal efficiency of PSII photochemistry (F_v/F_m), xanthophylls, and ABA in the dsm2 mutant suggest that the drought hypersensitivity of dsm2 is due to the combination of impairments in the xanthophyll cycle and ABA synthesis under drought stress conditions. DSM2 overexpression lines, possessing high F_v/F_m and NPQ, showed significantly improved drought resistance at both seedling and reproductive stages. Furthermore, our results imply that DSM2 may be the major member of the BCH family in rice for controlling zeaxanthin synthesis in response to dehydration stresses.