A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Biochemical characterization of rice xylan biosynthetic enzymes in determining xylan chain elongation and substitutions.

Grass xylan consists of a linear chain of ß-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or (methyl)glucuronic acid [(Me)GlcA] residues and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remain elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto Araf-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution pattern of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

A mechanism for hereditary angioedema caused by a methionine-379-to-lysine substitution in kininogens.

ABSTRACT: Hereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low-molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.

An Arabidopsis family GT106 glycosyltransferase is essential for xylan biosynthesis and secondary wall deposition.

MAIN CONCLUSION: We have demonstrated that the Arabidopsis FRA9 (fragile fiber 9) gene is specifically expressed in secondary wall-forming cells and essential for the synthesis of the unique xylan reducing end sequence. Xylan is made of a linear chain of ß-1,4-linked xylosyl (Xyl) residues that are often substituted with (methyl)glucuronic acid [(Me)GlcA] side chains and may be acetylated at O-2 and/or O-3. The reducing end of xylan from gymnosperms and dicots contains a unique tetrasaccharide sequence consisting of ß-D-Xylp-(1 → 3)-α-L-Rhap-(1 → 2)-α-D-GalpA-(1 → 4)-D-Xylp, the synthesis of which requires four different glycosyltransferase activities. Genetic analysis in Arabidopsis thaliana has so far implicated three glycosyltransferase genes, FRA8 (fragile fiber 8), IRX8 (irregular xylem 8) and PARVUS, in the synthesis of this unique xylan reducing end sequence. Here, we report the essential role of FRA9, a member of glycosyltransferase family 106 (GT106), in the synthesis of this sequence. The expression of the FRA9 gene was shown to be induced by secondary wall master transcriptional regulators and specifically associated with secondary wall-forming cells, including xylem and fiber cells. T-DNA knockout mutation of the FRA9 gene caused impaired secondary cell wall thickening in leaf veins and a severe arrest of plant growth. RNA interference (RNAi) downregulation of FRA9 led to a significant reduction in secondary wall thickening of fibers, a deformation of xylem vessels and a decrease in xylan content. Structural analysis of xylanase-released xylooligomers revealed that RNAi downregulation of FRA9 resulted in a diminishment of the unique xylan reducing end sequence and complete methylation of xylan GlcA side chains, chemotypes reminiscent of those of the fra8, irx8 and parvus mutants. Furthermore, two FRA9 close homologs from Populus trichocarpa were found to be wood-associated functional orthologs of FRA9. Together, our findings uncover a member of the GT106 family as a new player involved in the synthesis of the unique reducing end sequence of xylan.

Independent recruitment of glycosyltransferase family 61 members for xylan substitutions in conifers.

MAIN CONCLUSION: Several pine members of the gymnosperm-specific GT61 clades were demonstrated to be arabinosyltransferases and xylosyltransferases catalyzing the transfer of 2-O-Araf, 3-O-Araf and 2-O-Xyl side chains onto xylooligomer acceptors, indicating their possible involvement in Araf and Xyl substitutions of xylan in pine. Xylan in conifer wood is substituted at O-2 with methylglucuronic acid (MeGlcA) as well as at O-3 with arabinofuranose (Araf), which differs from xylan in dicot wood that is typically decorated with MeGlcA but not Araf. Currently, glycosyltransferases responsible for conifer xylan arabinosylation have not been identified. Here, we investigated the roles of pine glycosyltransferase family 61 (GT61) members in xylan substitutions. Biochemical characterization of four pine wood-associated GT61 members showed that they exhibited three distinct glycosyltransferase activities involved in xylan substitutions. Two of them catalyzed the addition of 2-O-α-Araf or 3-O-α-Araf side chains onto xylooligomer acceptors and thus were named Pinus taeda xylan 2-O-arabinosyltransferase 1 (PtX2AT1) and 3-O-arabinosyltransferase 1 (PtX3AT1), respectively. Two other pine GT61 members were found to be xylan 2-O-xylosyltransferases (PtXYXTs) adding 2-O-ß-Xyl side chains onto xylooligomer acceptors. Furthermore, sequential reactions with PtX3AT1 and the PtGUX1 xylan glucuronyltransferase demonstrated that PtX3AT1 could efficiently arabinosylate glucuronic acid (GlcA)-substituted xylooligomers and likewise, PtGUX1 was able to add GlcA side chains onto 3-O-Araf-substituted xylooligomers. Phylogenetic analysis revealed that PtX2AT1, PtX3AT1 and PtXYXTs resided in three gymnosperm-specific GT61 clades that are separated from the grass-expanded GT61 clade harboring xylan 3-O-arabinosyltransferases and 2-O-xylosyltransferases, suggesting that they might have been recruited independently for xylan substitutions in gymnosperms. Together, our findings have established several pine GT61 members as xylan 2-O- and 3-O-arabinosyltransferases and 2-O-xylosyltransferases and they indicate that pine xylan might also be substituted with 2-O-Araf and 2-O-Xyl side chains.

Identification of xylan arabinosyl 2-O-xylosyltransferases catalyzing the addition of 2-O-xylosyl residue onto arabinosyl side chains of xylan in grass species.

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

A mechanism for hereditary angioedema caused by a lysine 311-to-glutamic acid substitution in plasminogen.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions.

MAIN CONCLUSION: Multiple rice GT61 members were demonstrated to be xylan arabinosyltransferases (XATs) mediating 3-O-arabinosylation of xylan and the functions of XATs and xylan 2-O-xylosyltransferases were shown to be conserved in grass species. Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Araf) substitutions. In this report, we demonstrated that four previously uncharacterized, Golgi-localized glycosyltransferases residing in clade A or B of the rice GT61 family were able to mediate 3-O-arabinosylation of xylan when heterologously expressed in the Arabidopsis gux1/2/3 triple mutant. Biochemical characterization of their recombinant proteins established that they were xylan arabinosyltransferases (XATs) capable of transferring Araf residues onto xylohexaose acceptors, and thus they were named OsXAT4, OsXAT5, OsXAT6 and OsXAT7. OsXAT5 and the previously identified OsXAT2 were shown to be able to arabinosylate xylooligomers with a degree of polymerization of as low as 3. Furthermore, a number of XAT homologs from maize, sorghum, Brachypodium and switchgrass were found to exhibit activities catalyzing Araf transfer onto xylohexaose, indicating that they are XATs involved in xylan arabinosylation in these grass species. Moreover, we revealed that homologs of another GT61 member, xylan 2-O-xylosyltransferase (XYXT1), from these grass species could mediate 2-O-xylosylation of xylan when expressed in the Arabidopsis gux1/2/3 mutant. Together, our findings indicate that multiple OsXATs are involved in 3-O-arabinosylation of xylan and the functions of XATs and XYXTs are conserved in grass species.

A Single Xyloglucan Xylosyltransferase Is Sufficient for Generation of the XXXG Xylosylation Pattern of Xyloglucan.

Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1 and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1, but not AtXXT5, were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.

A Group of O-Acetyltransferases Catalyze Xyloglucan Backbone Acetylation and Can Alter Xyloglucan Xylosylation Pattern and Plant Growth When Expressed in Arabidopsis.

Xyloglucan is a major hemicellulose in plant cell walls and exists in two distinct types, XXXG and XXGG. While the XXXG-type xyloglucan from dicot species only contains O-acetyl groups on side-chain galactose (Gal) residues, the XXGG-type xyloglucan from Poaceae (grasses) and Solanaceae bears O-acetyl groups on backbone glucosyl (Glc) residues. Although O-acetyltransferases responsible for xyloglucan Gal acetylation have been characterized, the biochemical mechanism underlying xyloglucan backbone acetylation remains to be elucidated. In this study, we showed that recombinant proteins of a group of DUF231 members from rice and tomato were capable of transferring acetyl groups onto O-6 of Glc residues in cello-oligomer acceptors, indicating that they are xyloglucan backbone 6-O-acetyltransferases (XyBATs). We further demonstrated that XyBAT-acetylated cellohexaose oligomers could be readily xylosylated by AtXXT1 (Arabidopsis xyloglucan xylosyltransferase 1) to generate acetylated, xylosylated cello-oligomers, whereas AtXXT1-xylosylated cellohexaose oligomers were much less effectively acetylated by XyBATs. Heterologous expression of a rice XyBAT in Arabidopsis led to a severe reduction in cell expansion and plant growth and a drastic alteration in xyloglucan xylosylation pattern with the formation of acetylated XXGG-type units, including XGG, XGGG, XXGG, XXGG,XXGGG and XXGGG (G denotes acetylated Glc). In addition, recombinant proteins of two Arabidopsis XyBAT homologs also exhibited O-acetyltransferase activity toward cellohexaose, suggesting their possible role in mediating xyloglucan backbone acetylation in vivo. Our findings provide new insights into the biochemical mechanism underlying xyloglucan backbone acetylation and indicate the importance of maintaining the regular xyloglucan xylosylation pattern in cell wall function.

Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

A Novel Rice Xylosyltransferase Catalyzes the Addition of 2-O-Xylosyl Side Chains onto the Xylan Backbone.

Xylan is a major hemicellulose in both primary and secondary walls of grass species. It consists of a linear backbone of ß-1,4-linked xylosyl residues that are often substituted with monosaccharides and disaccharides. Xylosyl substitutions directly on the xylan backbone have not been reported in grass species, and genes responsible for xylan substitutions in grass species have not been well elucidated. Here, we report functional characterization of a rice (Oryza sativa) GT61 glycosyltransferase, XYXT1 (xylan xylosyltransferase1), for its role in xylan substitutions. XYXT1 was found to be ubiquitously expressed in different rice organs and its encoded protein was targeted to the Golgi, the site for xylan biosynthesis. When expressed in the Arabidopsis gux1/2/3 triple mutant, in which xylan was completely devoid of sugar substitutions, XYXT1 was able to add xylosyl side chains onto xylan. Glycosyl linkage analysis and comprehensive structural characterization of xylooligomers generated by xylanase digestion of xylan from transgenic Arabidopsis plants expressing XYXT1 revealed that the side chain xylosyl residues were directly attached to the xylan backbone at O-2, a substituent not present in wild-type Arabidopsis xylan. XYXT1 was unable to add xylosyl residues onto the arabinosyl side chains of xylan when it was co-expressed with OsXAT2 (Oryza sativa xylan arabinosyltransferase2) in the gux1/2/3 triple mutant. Furthermore, we showed that recombinant XYXT1 possessed an activity transferring xylosyl side chains onto xylooligomer acceptors, whereas recombinant OsXAT2 catalyzed the addition of arabinosyl side chains onto xylooligomer acceptors. Our findings from both an in vivo gain-of-function study and an in vitro recombinant protein activity assay demonstrate that XYXT1 is a novel ß-1,2-xylosyltransferase mediating the addition of xylosyl side chains onto xylan.

Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment.

The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.

In vitro release properties of encapsulated blueberry (Vaccinium ashei) extracts.

We aimed to determine the effect of encapsulation on the release properties of blueberry extracts during simulated gastrointestinal digestion. An ethanolic pomace extract was microencapsulated with whey protein isolate via spray drying. The in vitro release of monomeric anthocyanins, phenolics and ferric reducing antioxidant activity of the microcapsules (W) were evaluated for the microcapsules and two non-encapsulated systems: ethanolic pomace extract (P) and freeze-dried juice (F). Concentrations of anthocyanin and phenolics were normalised prior to digestion. Results showed that antioxidant activity was in the order of: F>W>P. Regardless of encapsulation, more phenolics were released from W and P than F. Anthocyanin concentration decreased after intestinal digestion for W, but remained constant for P and F. MALDI-MS showed similar spectra for P and F but not for W. The spray-dried product has comparable release characteristics to freeze-dried juice, and may be investigated for food applications.

Evidence for no sexual isolation between Drosophila albomicans and D. nasuta.

Sexual isolation, the reduced tendency to mate, is one of the reproductive barriers that prevent gene flow between different species. Various species-specific signals during courtship contribute to sexual isolation between species. Drosophila albomicans and D. nasuta are closely related species of the nasuta subgroup within the Drosophila immigrans group and are distributed in allopatry. We analyzed mating behavior and courtship as well as cuticular hydrocarbon profiles within and between species. Here, we report that these two species randomly mated with each other. We did not observe any sexual isolation between species or between strains within species by multiple-choice tests. Significant difference in the courtship index was detected between these two species, but males and females of both species showed no discrimination against heterospecific partners. Significant quantitative variations in cuticular hydrocarbons between these two species were also found, but the cuticular hydrocarbons appear to play a negligible role in both courtship and sexual isolation between these two species. In contrast to the evident postzygotic isolation, the lack of sexual isolation between these two species suggests that the evolution of premating isolation may lag behind that of the intergenomic incompatibility, which might be driven by intragenomic conflicts.

Sexual isolation between North American and Bogota strains of Drosophila pseudoobscura.

Sexual isolation, the reduced ability of organisms of different species to successfully mate, is one of the reproductive barriers that prevent gene flow between different taxa. Various species-specific signals during courtship are attributed to the sexual isolation between them. Drosophila pseudoobscura has been widely used to study the behavioral and genetic mechanisms underlying selection for sexual isolation, as a model system for speciation. D. pseudoobscura and its sibling species, D. persmilis, live together in many locations but are reproductively isolated from one another. North American geographic strains of D. pseudoobscura from the American West mate at random. Several decades ago, D. pseudoobscura was collected in the vicinity of Bogota, Colombia, and later named the subspecies D. pseudoobscura bogotana. Nearly 5,000 matings were observed in this study. We analyzed mating behavior and cuticular hydrocarbon profiles as well as courtship within and between North American and Bogota strains of D. pseudoobscura. Here we report for the first time that Bogota strains of D. pseudoobscura do show statistically significant sexual isolation from North American strains. In addition, there are quantitative variations in cuticular hydrocarbons as well as in courtship behavior between Bogota and North American strains, and females of both North American and Bogota strains show strong preference for North American strain males having high mating propensities, suggesting that the Bogota strains are at an early stage that could lead to a separate species.

Determination of the degradation products of selected sulfonated phenylazonaphthol dyes treated by white rot fungus Pleurotus ostreatus by capillary electrophoresis coupled with electrospray ionization ion trap mass spectrometry.

The removal of water-soluble sulphonated phenylazonaphthol dye effluents generated by textile industries is an important issue in wastewater treatment. Microbial treatment of environmental pollutants including dyes, with white rot fungi has received wide attention as a potential alternative for conventional methods in wastewater treatment. Three sulphonated phenylazonaphthol dyes with similar molecular structures Acid Orange 7, Acid Orange 8 and Mordant Violet 5 were selected and degraded by the white rot fungus Pleurotus ostreatus. Chemical instrumental analysis methods such as high-performance liquid chromatography (HPLC) and capillary electrophoresis combined with electrospray ionization mass spectrometry (CE-ESI-MS) were used to identify the degraded dyes. Mordant Violet 5 had two degradation pathways when degraded by P. ostreatus. The first degradation pathway for Mordant Violet 5 was for trans structure and the cis-Mordant Violet 5 followed the second pathway. Acid Orange 8 and Acid Orange 7 had the same degradation mechanism as the first degradation mechanism for Mordant Violet 5, that is cleavage of azo bond at the naphthalene ring side where benzenesulfonic acid and 1,2-naphthoquinone are formed.

Study of biodegradation products from azo dyes in fungal degradation by capillary electrophoresis/electrospray mass spectrometry.

Biodegradation products from four model sulfonated azo dyes Orange II, Acid Orange 8, Food Yellow 3, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonic acid, sodium salt (4HABA), during fungal degradation were determined by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) with electrospray ionization and a coaxial sheath flow interface. The development and optimization of this analytical method including the sheath liquid composition and flow rate, nebulizing gas flow rate, carrier electrolyte, and MS voltage are described herein. Detection of unknown biodegradation products was carried out under negative ion mode with base peak electrophorogram (BPE) or extractive ion electrophorogram (EIE) monitoring. A volatile ammonium acetate buffer (10 mM) without organic modifier and a shealth liquid made from 2-propanol and water (80:20, v/v) were suited for the separation and ESI interface. The sulfonated ion was the base peak for model azo dyes and their metabolites containing sulfonic group. Results showed that the tested azo dyes were degraded quickly in the culture of white rot fungus, Pleurotus ostreatus in 3 days with the major biodegradation products being 4-hydroxy-benzenesulfonic acid, 3-methyl-4-hydroxy-benzenesulfonic acid, benzenesulfonic acid, 1,2-naphthoquinone-6-sulfonic acid and 3-methyl-benzenesulfonic acid.

Developmental isolation and subsequent adult behavior of Drosophila paulistorum. VI. Quantitative variation in cuticular hydrocarbons.

Our previous studies have demonstrated that long-term chemical contact with heterospecifics during development minimized unproductive heterospecific matings among the six Drosophila paulistorum semispecies. When socially isolated from conspecifics, discrimination significantly decreased so that more ultimately unproductive heterospecific matings occurred. Such results suggest that learning and social experiences play roles in mate recognition, using chemical information. In investigations into the development of discriminatory behavior in D. paulistorum, social experiences influence the production of cuticular hydrocarbons in both sexes among the six different semispecies of D. paulistorum. Produced by both sexes, 2-methyl triacontane has been detected from egg stages on, gradually increasing with age. However, 11-docosenyl acetate, a male pheromone, was not produced until early adult stages. There were consistent significant differences in quantities and ratios of each of these hydrocarbons among the six semispecies, contributing to their reproductive isolation. Furthermore, we found significant quantitative differences in hydrocarbons between socially isolated flies and communally raised ones: Socially isolated flies produced significantly greater quantities of hydrocarbons than communally raised flies, consistent with previous behavioral data, as isolated flies court more vigorously and often.