Herbivore-Induced SABATH Methyltransferases of Maize That Methylate Anthranilic Acid Using S-Adenosyl-l-Methionine

Volatile methyl esters are common constituents of plant volatiles with important functions in plant defense. To study the biosynthesis of these compounds, especially methyl anthranilate and methyl salicylate, we identified a group of methyltransferases that are members of the SABATH enzyme family in maize (Zea mays). In vitro biochemical characterization after bacterial expression revealed three S-adenosyl-l-methionine-dependent methyltransferases with high specificity for anthranilic acid as a substrate. Of these three proteins, Anthranilic Acid Methyltransferase1 (AAMT1) appears to be responsible for most of the S-adenosyl-l-methionine-dependent methyltransferase activity and methyl anthranilate formation observed in maize after herbivore damage. The enzymes may also be involved in the formation of low amounts of methyl salicylate, which are emitted from herbivore-damaged maize. Homology-based structural modeling combined with site-directed mutagenesis identified two amino acid residues, designated tyrosine-246 and glutamine-167 in AAMT1, which are responsible for the high specificity of AAMTs toward anthranilic acid. These residues are conserved in each of the three main clades of the SABATH family, indicating that the carboxyl methyltransferases are functionally separated by these clades. In maize, this gene family has diversified especially toward benzenoid carboxyl methyltransferases that accept anthranilic acid and benzoic acid.Volatile compounds have important roles in the reproduction and defense of plants. Volatiles can attract pollinators and seed dispersers (Dobson and Bergström, 2000; Knudsen et al., 2006) or function as indirect defense compounds that attract natural enemies of herbivores (Dicke, 1994; Degenhardt et al., 2003; Howe and Jander, 2008). A well-studied example for the role of volatiles in plant defense is the tritrophic interaction between maize (Zea mays) plants, their lepidopteran herbivores, and parasitoid wasps of the herbivores. After damage by larvae of Spodoptera species, maize releases a complex volatile blend containing different classes of natural products (Turlings et al., 1990; Turlings and Benrey, 1998a). This volatile blend can be used as a cue by parasitic wasps to find hosts for oviposition (Turlings et al., 1990, 2005). After parasitization, lepidopteran larvae feed less and die upon emergence of the adult wasp, resulting in a considerable reduction in damage to the plant (Hoballah et al., 2002, 2004). The composition of the maize volatile blend is complex, consisting of terpenoids and products of the lipoxygenase pathway, along with three aromatic compounds: indole, methyl anthranilate, and methyl salicylate (Turlings et al., 1990; Degen et al., 2004; Köllner et al., 2004a). In the last decade, several studies have addressed the biosynthesis of terpenoids (Shen et al., 2000; Schnee et al., 2002, 2006; Köllner et al., 2004b, 2008a, 2008b) and indole (Frey et al., 2000, 2004) in maize. The formation of methyl anthranilate and methyl salicylate, however, has not been elucidated.Methyl anthranilate and methyl salicylate are carboxyl methyl esters of anthranilic acid, an intermediate of Trp biosynthesis, and the plant hormone salicylic acid, respectively. Our understanding of methyl anthranilate biosynthesis in plants is very limited. The only enzyme that has been described to be involved in methyl anthranilate synthesis is the anthraniloyl-CoA:methanol acyltransferase in Washington Concord grape (Vitis vinifera; Wang and De Luca, 2005). In contrast, the biosynthesis of methyl salicylate has been well studied in several plant species, such as Clarkia brewerii (Ross et al., 1999), Arabidopsis (Arabidopsis thaliana; Chen et al., 2003), and rice (Oryza sativa; Xu et al., 2006; Koo et al., 2007; Zhao et al., 2010). In all these species, methyl salicylate is synthesized by the action of S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase (SAMT). The apparent homology of SAMTs from different plant species suggests that methyl salicylate formation in maize, a species closely related to rice, is also catalyzed by an SAMT. SAMT enzymes are considered part of a larger family of methyltransferases called SABATH methyltransferases (D''Auria et al., 2003). The SABATH family also includes methyltransferases producing other methyl esters such as methyl benzoate, methyl jasmonate, and methyl indole-3-acetate (Seo et al., 2001; Effmert et al., 2005; Qin et al., 2005; Song et al., 2005; Zhao et al., 2007). An activity forming methyl anthranilate has not been described in the SABATH family, despite the striking structural similarity between methyl anthranilate and methyl salicylate or methyl benzoate. Two different classes of enzymes, methanol acyl transferases and methyltransferases, therefore, might be responsible for methyl anthranilate biosynthesis in maize (Fig. 1). Some of the SABATH methyltransferases have been shown previously to have methyltransferase activity in vitro using anthranilic acid as substrate (Chen et al., 2003; Zhao et al., 2010), but the biological relevance of such activity is unknown.Open in a separate windowFigure 1.The biosynthesis of methyl anthranilate from anthranilic acid can proceed over two pathways. Pathway A has been documented in grape, while pathway B is demonstrated here. AMAT, Anthraniloyl-CoA:methanol acyltransferase; SAH, S-adenosyl-l-homocysteine.In our ongoing attempt to investigate the biosynthesis and function of maize volatiles, we have studied the biosynthesis of the aromatic methyl esters, methyl salicylate and methyl anthranilate, and their regulation by herbivory. Biochemical characterization of maize benzenoid carboxyl methyltransferases of the SABATH family led to the discovery of a group of anthranilic acid methyltransferases (AAMTs). Homology-based structural modeling combined with site-directed mutagenesis identified the residues critical for the binding of the anthranilic acid substrate. Such functionally important residues are responsible for the diversification and evolution of benzenoid carboxyl methyltransferases in plants.     

Studies of ent-kaurane diterpenes from Oyedaea verbesinoides for their inhibitory activity on vascular smooth muscle contraction

From the aerial parts of Oyedaea verbesinoides nine ent-kauranes and a sesquiterpene were isolated. ent-9alpha-Hydroxy-kaur-16-en-19-oic acid, ent-15beta-tigloyloxy-9alpha-hydroxy-kaur-16-en-19-oic acid, ent-15beta-angeloyloxy-9alpha-hydroxy-kaur-16-en-19-oic acid, ent-16alpha-hydroxykaurane and 1alpha-angeloyloxy-carotol are new for the genus or the species and ent-15beta-angeloyloxy-7alpha,9alpha-dihydroxy-kaur-16-en-19-oic acid is reported for the first time. Structure elucidation was based on one and two dimensional NMR as well as ESI and CI-MS analysis. Some diterpenes were proven to exhibit inhibitory effects on smooth muscle contraction on rat aorta.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

From a Traditional Medicinal Plant to a Rational Drug: Understanding the Clinically Proven Wound Healing Efficacy of Birch Bark Extract

Background

Birch bark has a long lasting history as a traditional medicinal remedy to accelerate wound healing. Recently, the efficacy of birch bark preparations has also been proven clinically. As active principle pentacyclic triterpenes are generally accepted. Here, we report a comprehensive study on the underlying molecular mechanisms of the wound healing properties of a well-defined birch bark preparation named as TE (triterpene extract) as well as the isolated single triterpenes in human primary keratinocytes and porcine ex-vivo wound healing models.

Methodology/Principal Findings

We show positive wound healing effects of TE and betulin in scratch assay experiments with primary human keratinocytes and in a porcine ex-vivo wound healing model (WHM). Mechanistical studies elucidate that TE and betulin transiently upregulate pro-inflammatory cytokines, chemokines and cyclooxygenase-2 on gene and protein level. For COX-2 and IL-6 this increase of mRNA is due to an mRNA stabilizing effect of TE and betulin, a process in which p38 MAPK and HuR are involved. TE promotes keratinocyte migration, putatively by increasing the formation of actin filopodia, lamellipodia and stress fibers. Detailed analyses show that the TE components betulin, lupeol and erythrodiol exert this effect even in nanomolar concentrations. Targeting the actin cytoskeleton is dependent on the activation of Rho GTPases.

Conclusion/Significance

Our results provide insights to understand the molecular mechanism of the clinically proven wound healing effect of birch bark. TE and betulin address the inflammatory phase of wound healing by transient up-regulation of several pro-inflammatory mediators. Further, they enhance migration of keratinocytes, which is essential in the second phase of wound healing. Our results, together with the clinically proven efficacy, identify birch bark as the first medical plant with a high potential to improve wound healing, a field which urgently needs effective remedies.     

Bioconversion of α-Damascone by Botrytis cinerea

Bioconversion of α-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-α-damascone, cis- and trans-3-hydroxy-α-damascone, γ-damascenone, 3-oxo-8, 9-dihydro-α-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-α-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-α-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.     

Inhibin/activin subunits alpha, beta-A and beta-B are differentially expressed in normal human endometrium throughout the menstrual cycle

Inhibins are dimeric glycoproteins composed of an alpha () subunit and one of two possible beta (-) subunits (A or B). The aims of this study were to assess the frequency and tissue distribution patterns of the inhibin subunits in normal human endometrium. Samples from human endometrium from proliferative phase (PP; n=32), early secretory phase (ES; n=10) and late secretory phase (LS; n=12) were obtained. Immunohistochemistry, immunofluorescence and a statistical analysis were performed. All three inhibin subunits were expressed by normal endometrium by immunohistochemistry and immunofluorescence. Inhibin- was primarily detected in glandular epithelial cells, while inhibin- subunits were additionally localised in stromal tissue. Inhibin- staining reaction increased significantly between PP and ES (P<0.05), PP and LS (P<0.01), and ES and LS (P<0.02). Inhibin-A and -B were significant higher in LS than PP (P<0.05) and LS than ES (P<0.05). All three inhibin subunits were expressed by human endometrium varying across the menstrual cycle. This suggests substantial functions in human implantation of inhibin- subunit, while stromal expression of the subunits could be important in the paracrine signalling for adequate endometrial maturation. The distinct expression in human endometrial tissue suggests a synthesis of inhibins into the lumen and a predominant secretion of activins into the stroma.I. Mylonas and U. Jeschke contributed equally to this work     

Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.     

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.     

Age-related changes in ovarian morphology of the South American tamarin Saguinus fuscicollis (Callitrichidae)

The aim of this histological study was to investigate the postnatal ontogeny of the ovaries in Saguinus fuscicollis to provide a detailed knowledge of the ovarian morphology for further endocrinological studies. Gonads from 43 animals between one day and 18 years of age were investigated. Based on the available material the ovarian development is characterized by seven distinct stages. 1. Neonatal stage : Folliculogenesis is still in progress. The ovarian medulla is filled with an intraovarian rete system, which forms open connections between the rete tubules and the cords containing oocytes. 2. Infantile stage : One month after birth the ovarian cortex is mainly composed of primordial follicles and a few primary follicles at the corticomedullary border, which are separated from each other by connective tissue. At two months, folliculogenesis is nearly completed. The first secondary follicles are present. 3. Juvenile stage : In six-month-old females weighing 184±9.5 g, folliculogenesis has reached the stage of secondary follicles with up to six layers of granulosa cells. In females of the same age weighing 251.5±37 g, antral follicles attain a maximum diameter of 925 μm m. 4. Pubertal stage : At the age of 8–10 months, corpora lutea accessoria (CLA) begin to form from atretic antral follicles. 5. Adult stage : The ovaries of all females older than 1.2 years are filled with large patches of interstitial gland tissue (IGT). In breeding females up to 58.4%of the antral follicles are intact. In non-breeding daughters living in the family group only 1.8%are intact, the rest are in various stages of atresia and form CLA. 6. Climacterial stage : With increasing age (8–13 years), the number of intact follicles decreases dramatically and IGT fills nearly the whole ovary. 7. Senile stage : In females older than 14 years, nearly all remaining follicles show signs of atresia.