Preen gland microbiota covary with major histocompatibility complex genotype in a songbird.

Pathogen-mediated selection at the major histocompatibility complex (MHC) is thought to promote MHC-based mate choice in vertebrates. Mounting evidence implicates odour in conveying MHC genotype, but the underlying mechanisms remain uncertain. MHC effects on odour may be mediated by odour-producing symbiotic microbes whose community structure is shaped by MHC genotype. In birds, preen oil is a primary source of body odour and similarity at MHC predicts similarity in preen oil composition. Hypothesizing that this relationship is mediated by symbiotic microbes, we characterized MHC genotype, preen gland microbial communities and preen oil chemistry of song sparrows (Melospiza melodia). Consistent with the microbial mediation hypothesis, pairwise similarity at MHC predicted similarity in preen gland microbiota. Counter to this hypothesis, overall microbial similarity did not predict chemical similarity of preen oil. However, permutation testing identified a maximally predictive set of microbial taxa that best reflect MHC genotype, and another set of taxa that best predict preen oil chemical composition. The relative strengths of relationships between MHC and microbes, microbes and preen oil, and MHC and preen oil suggest that MHC may affect host odour both directly and indirectly. Thus, birds may assess MHC genotypes based on both host-associated and microbially mediated odours.

Food stress, but not experimental exposure to mercury, affects songbird preen oil composition.

Mercury is a global pollutant and potent neurotoxic metal. Its most toxic and bioavailable form, methylmercury, can have both lethal and sublethal effects on wildlife. In birds, methylmercury exposure can disrupt behavior, hormones, the neuroendocrine system, and feather integrity. Lipid-rich tissues and secretions may be particularly susceptible to disruption by lipophilic contaminants such as methylmercury. One such substance is feather preen oil, a waxy secretion of the uropygial gland that serves multiple functions including feather maintenance, anti-parasitic defense, and chemical signaling. If methylmercury exposure alters preen oil composition, it could have cascading effects on feather quality, susceptibility to ectoparasites, and mate choice and other social behaviors. We investigated whether exposure to methylmercury, either alone or in association with other stressors, affects preen oil chemical composition. We used a two-factor design to expose adult song sparrows (Melospiza melodia) to an environmentally relevant dietary dose of methylmercury and/or to another stressor (unpredictable food supply) for eight weeks. The wax ester composition of preen oil changed significantly over the 8-week experimental period. This change was more pronounced in the unpredictable food treatment, regardless of dietary methylmercury. Contrary to our prediction, we found no main effect of methylmercury exposure on preen oil composition, nor did methylmercury interact with unpredictable food supply in predicting the magnitude of chemical shifts in preen oil. While it remains critical to study sublethal effects of methylmercury on wildlife, our findings suggest that the wax ester composition of preen oil is robust to environmentally relevant doses of this contaminant.

Chemical composition of preen wax reflects major histocompatibility complex similarity in songbirds.

In jawed vertebrates, genes of the major histocompatibility complex (MHC) play a key role in immunity by encoding cell-surface proteins that recognize and bind non-self antigens. High variability at MHC suggests that these loci may also function in social signalling such as mate choice and kin recognition. This requires that MHC genotype covaries with some perceptible phenotypic trait. In mammals and fish, MHC is signalled chemically through volatile and non-volatile peptide odour cues, facilitating MHC-dependent mate choice and other behaviours. In birds, despite evidence for MHC-dependent mating, candidate mechanisms for MHC signalling remain largely unexplored. However, feather preen wax has recently been implicated as a potential source of odour cues. We examined whether the chemical composition of preen wax correlates with MHC class IIß genotypes of wild song sparrows (Melospiza melodia). Pairwise chemical distance reflected amino acid distance at MHC for male-female dyads, although not for same-sex dyads. Chemical diversity did not reflect MHC diversity. We used gas chromatography-mass spectrometry (GC-MS) to characterize preen wax compounds, and identified four wax esters that best reflect MHC similarity. Provided songbirds can detect variation in preen wax composition, this cue may allow individuals to assess MHC compatibility of potential mates.

Dietary carotenoid levels affect carotenoid and retinoid allocation in female Chinook salmon Oncorhynchus tshawytscha.

This study examined the effect of dietary carotenoid availability on carotenoid and retinoid concentrations in the flesh, plasma, skin and eggs of female Chinook salmon Oncorhynchus tshawytscha. Carotenoid concentrations in all tissues were closely related to dietary availability. Early in the breeding season, carotenoids were stored primarily in the muscle, with a flesh carotenoid concentration of 9.9 microg g(-1) in fish fed a high carotenoid diet compared with 1.9 microg g(-1) in fish fed a low carotenoid diet. During the breeding season, carotenoid reserves were mobilized predominantly to the eggs and also to the skin. By the end of the breeding season, carotenoid concentrations in the eggs were 17.9 microg g(-1) in fish fed a high carotenoid diet and 3.9 microg g(-1) in fish fed a low carotenoid diet. Conversely, egg retinoid concentrations were only c. 20% lower in fish fed a low v. high carotenoid diet, which suggests that retinoid concentrations were not limited by the availability of carotenoid precursors. Egg carotenoid concentrations were not correlated with either skin carotenoid concentration or colouration, which suggests that female carotenoid displays are not a reliable signal that males can use to evaluate egg carotenoid resources.

Isolation of an antimicrobial compound from Impatiens balsamina L. using bioassay-guided fractionation.

By using brine shrimp (Artemia salina) lethality test-guided fractionation, a single bioactive compound (LC(50)=26 ppm) was isolated from the 95% ethanol extract of the dried aerial parts of Impatiens balsamina L. and subsequently identified as 2-methoxy-1,4-naphthoquinone (MNQ). The structure of MNQ was confirmed by UV, FT-IR, MS, and 1-and 2-D NMR spectroscopy. The antimicrobial activity of MNQ was evaluated using 12 bacterial and eight fungal strains. Five gram-positive and two gram-negative bacteria as well as all eight fungi (including multi-drug resistant strains) tested were highly sensitive to MNQ. A tea prepared according to traditional methods was found to contain sufficient MNQ to account for its antimicrobial properties.

The poly(phenolic) domain of potato suberin: a non-lignin cell wall bio-polymer.

Suberized plant cell walls have three distinguishing features: (1) tissue specificity, (2) a poly(aliphatic) domain and (3) a unique, "lignin-like" poly(phenolic) domain. With respect to the latter, comparisons have often been made to lignin, but the unique phenolic composition of suberized cells yields a unique polymer better designated as a poly(phenolic) domain. Potato tubers that have been induced to suberize through wounding make an excellent model system with which the chemistry, biochemistry and macromolecular assembly of the suberin poly(phenolic) domain can be monitored. For example, wound healing potato tubers have been used to determine the unique hydroxycinnamic acid nature of its poly(phenolic) domain using specific carbon-13 labeling studies and specific chemical degradation techniques (e.g. thioacidolysis). Furthermore, a suberization-associated anionic peroxidase has been purified from suberizing potato tubers and subsequently shown to oxidize hydroxycinnamic acids (and their derivatives) in preference to monolignols, as well as yield an unique polymer in vitro. We have since extended these studies to begin analyzing the macromolecular assembly process leading to the deposition of this suberized tissue specific domain. To this end we have begun to describe an H(2)O(2)-generating system with NAD(P)H-dependent oxidase-like properties that is temporally associated with the formation of potato suberin poly(phenolics) during suberization. Herein we describe our progress to date.

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis.

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.

Biochemical characterization of the suberization-associated anionic peroxidase of potato.

The anionic peroxidase associated with the suberization response in potato (Solanum tuberosum L.) tubers during wound healing has been purified and partially characterized at the biochemical level. It is a 45-kD, class III (plant secretory) peroxidase that is localized to suberizing tissues and shows a preference for feruloyl (o-methoxyphenol)-substituted substrates (order of substrate preference: feruloyl > caffeoyl > p-coumaryl approximately syringyl) such as those that accumulate in tubers during wound healing. There was little influence on oxidation by side chain derivatization, although hydroxycinnamates were preferred over the corresponding hydroxycinnamyl alcohols. The substrate specificity pattern is consistent with the natural substrate incorporation into potato wound suberin. In contrast, the cationic peroxidase(s) induced in response to wound healing in potato tubers is present in both suberizing and nonsuberizing tissues and does not discriminate between hydroxycinnamates and hydroxycinnamyl alcohols. A synthetic polymer prepared using E-[8-(13)C]ferulic acid, H(2)O(2), and the purified anionic enzyme contained a significant amount of cross-linking through C-8, albeit with retention of unsaturation.

The macromolecular aromatic domain in suberized tissue: a changing paradigm.

As a structural feature of specialized cell walls, suberization remains an enigma, despite its obvious importance both during normal growth and development and as a stress response in plants. While it is clear that suberized tissues contain both polyaromatic and polyaliphatic domains, and that each of these has its own unique characteristics, whether there is a contiguous macromolecule that can be called suberin is an open question. From a structural perspective, the aromatic domain is unique and distinct from lignin, and is apparently comprised primarily of (poly)hydroxycinnamates, such as amides (e.g., feruloyltyramine). The aliphatic domain is also unique, being quite distinct from cutin in terms of both its chemical composition and cellular location. In the present paper, histochemical, structural and biochemical data, particularly, regarding the polyaromatic domain of suberized tissues, are critically reviewed. A revised description of the polyaromatic domain of suberized tissues, based on the consensus that is emerging from the current data, is presented and especially includes a spatially distinct (poly)hydroxycinnamoyl-containing macromolecule.

Towards the specification of consecutive steps in macromolecular lignin assembly.

When Pinus taeda cell suspension cultures are exposed to 8% sucrose solution, the cells undergo significant intracellular disruption, irregular wall thickening/lignification with concomitant formation of an 'extracellular lignin precipitate. However, addition of potassium iodide (KI), an H202 scavenger, inhibits this lignification response, while the ability to synthesize the monolignols, p-coumaryl and coniferyl alcohols, is retained. Lignin synthesis (i.e. polymerization) is thus temporarily correlated with H202 generation, strongly implying a regulatory role for the latter. Time course analyses of extracellular metabolites leading up to polymer formation reveal that coniferyl alcohol, but not p-coumaryl alcohol, undergoes substantial coupling reactions to give various lignans. Of these, the metabolites, dihydrodehydrodiconiferyl alcohol, shonanin (divanillyl tetrahydrofuran) and its apparent aryl tetralin derivative, cannot be explained simply on the basis of phenolic coupling. It is proposed that these moieties are the precursors of so-called reduced substructures in the lignin macromolecule. This adds a new perspective to the lignin assembly mechanism.

Hydroxycinnamic acid-derived polymers constitute the polyaromatic domain of suberin.

Suberin is an abundant, complex, intractable, plant cell wall polymeric network that forms both protective and wound-healing layers. Its function is, therefore, critical to the survival of all vascular plants. Its chemical structure and biosynthesis are poorly defined, although it is known to consist of both aromatic and aliphatic domains. While the composition of the aliphatic component has been fairly well characterized, that of the phenolic component has not. Using a combination of specific carbon-13 labeling techniques, and in situ solid state 13C NMR spectroscopic analysis, we now provide the first direct evidence for the nature of the phenolic domain of suberin and report here that it is almost exclusively comprised of a covalently linked, hydroxycinnamic acid-derived polymeric matrix.

Lignification in cell suspension cultures of Pinus taeda. In situ characterization of a gymnosperm lignin.

Pinus taeda suspension cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed only primary cell wall formation and essentially no lignification, as determined by histochemical, ultrastructural, chemical, and NMR spectroscopic analyses. However, these cultures maintained a functional phenylpropanoid pathway as demonstrated by formation of the lignans (-)-matairesinol and (-)-pinoresinol. Administration of [1-13C]Phe to these cultures, followed by solid-state carbon-13 NMR spectral analysis of their cell walls, demonstrated that the phenylalanine incorporated into the cell wall matrix was primarily as protein, rather than lignin. Successive transfer of the 2,4-dichlorophenoxyacetic acid-grown cultures to alpha-naphthaleneacetic acid-containing medium induced cell wall thickening concomitant with lignification. The presence of lignin was confirmed by histochemical, ultrastructural, chemical, biochemical, and NMR spectroscopic analyses. Specific labeling of the lignin polymer in situ with [1-13C]-, [2-13C]-, and [3-13C]Phe and analysis of the cell wall preparations by solid-state carbon-13 NMR spectroscopy permitted the first direct determination of the in situ bonding patterns in a gymnosperm lignin. Several dominant interunit linkages were observed, including beta-O-aryl, furanofuran, phenylcoumarin, and phenolic-linked monolignols, consistent with those predicted but hitherto not proven. Finally, milled wood lignin derivatives prepared from these 13C-specifically enriched lignin tissues gave a relatively high fidelity copy of the native lignin.

Alkyl ferulates in wound healing potato tubers.

Seven ferulic acid esters of 1-alkanols ranging in carbon length from C16 to C28 were synthesized and an HPLC protocol for their separation developed. Extracts prepared from wound healing potato (Solanum tuberosum) tubers and analysed by HPLC indicated that alkyl ferulate esters begin to accumulate 3-7 days after wound treatment. Of the nine esters identified by EIMS, (including two esters of odd chain length alkanols) hexadecyl and octadecyl ferulates were predominant. Alkyl ferulate esters were restricted to the wound periderm.

Phenylalanine Ammonia-Lyase from Tomato Cell Cultures Inoculated with Verticillium albo-atrum.

Tomato (Lycopersicon esculentum Mill.) cell suspension cultures accumulated wall-bound phenolic materials in response to inoculation with Verticillium albo-atrum Reinke et Berth. in a fashion analogous to that observed in whole plants. Both monomeric and polymeric materials were recovered. Deposition of phenolics into the cell walls of inoculated tomato cell cultures was inhibited by the phenylalanine ammonia-lyase (PAL) inhibitor, 2-amino-2-indanephosphate. Tomato PAL activity was induced over 12-fold by fungal inoculation, with a concomitant increase in the corresponding mRNA. The enzyme was purified >3400-fold, to apparent homogeneity, by anion-exchange chromatography, chromatofocusing, and gel filtration. The holoenzyme had a molecular mass of 280 to 320 kilodaltons, comprising 74-kilodalton subunits, and displayed an isoelectric point of 5.6 to 5.7. Induced PAL displayed apparent Michaelis-Menten kinetics (K(m) = 116 micromolar) and was not appreciably inhibited by its product cinnamic acid. Chromatographic analysis did not reveal multiple forms of the enzyme in either inoculated or uninoculated cultures.