An assessment of the antibacterial activity in larval excretion/secretion of four species of insects recorded in association with corpses, using Lucilia sericata Meigen as the marker species.

The relative antibacterial activities of excretion/secretion (ES) from two carrion-feeding insects, Calliphora vicina Robineau-Desvoidy and Dermestes maculatus DeGeer, and a detritivore, Tenebrio molitor Linnaeus, were compared to that of Lucilia sericata Meigen, a species with ES of known antibacterial capacity, in order to explore the antimicrobial potential of other carrion and detritivore species. Viable counts were used to assess time-kill of ES against five bacterial species, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Proteus mirabilis. Antibacterial activity was recorded in all four insect species although T. molitor and D. maculatus were the most effective in controlling growth of P. mirabilis. The blowflies were more effective in controlling a wider range of both Gram-positive and Gram-negative bacteria. The larval ES from all species was shown to reduce bacterial growth rate although differences in antibacterial spectrum were noted and the degree of potency varied between the four species. These differences may be explained ecologically by the different colonisation times of each insect species on the corpse. Overall, this study demonstrates that research into other carrion-feeding insect species has potential to provide an increased source of antimicrobial chemicals to broaden the range of bacterial species beyond that currently controlled using L. sericata.

Transcriptional activation of plant defense genes.

Significant progress has been made in the characterization of disease resistance genes and receptors for pathogen avirulence signals and non-specific elicitors. Some components involved in elicitor-induced signal transduction have been identified. Phosphorylation of transcription factors has been found to be one of the mechanisms regulating their cellular localization, DNA binding and transcription activities for defense gene activation.

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory.

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

Signal transduction in plant immunity.

Significant recent advances in the understanding of plant defense mechanisms include the isolation and characterization of resistance genes against bacterial, fungal and viral pathogens, the identification of genes involved in cell death, and the demonstration of the involvement of reactive oxygen species and salicylic acid in the signal-transduction pathways for expression of induced resistance.

CUE1: A Mesophyll Cell-Specific Positive Regulator of Light-Controlled Gene Expression in Arabidopsis.

Light plays a key role in the development and physiology of plants. One of the most profound effects of light on plant development is the derepression of expression of an array of light-responsive genes, including the genes encoding the chlorophyll a/b binding proteins (CAB) of photosystem II. To understand the mechanism by which light signals nuclear gene expression, we developed a genetic selection to identify mutants with reduced CAB transcription. Here, we describe a new Arabidopsis locus, CUE1 (for CAB underexpressed). Mutations at this locus result in defects in expression of several light-regulated genes, specifically in mesophyll but not in bundle-associated or epidermis cells. Reduced accumulation of CAB and other photosynthesis-related mRNAs in the mesophyll was correlated with defects in chloroplast development in these cells, resulting in a reticulate pattern with veins greener than the interveinal regions of leaves. Moreover, chalcone synthase mRNA, although known to be regulated by both phytochrome and a blue light receptor, accumulated normally in the leaf epidermis. Dark basal levels of CAB expression were unaffected in etiolated cue1 seedlings; however, induction of CAB transcription by pulses of red and blue light was reduced, suggesting that CUE1 acts downstream from both phytochrome and blue light photoreceptors. CUE1 appears to play a role in the primary derepression of mesophyll-specific gene expression in response to light, because cue1 mutants are severely deficient at establishing photoautotrophic growth. Based on this characterization, we propose that CUE1 is a cell-specific positive regulator linking light and intrinsic developmental programs in Arabidopsis leaf mesophyll cells.

Functional organization of the simian virus 40 origin of DNA replication.

To define the sequence elements involved in initiation of DNA synthesis at the simian virus 40 origin of replication, we determined the relative replication efficiencies in vitro and in vivo of templates containing a variety of mutations within the origin region. Replication of the mutants in vitro was assayed by the cell-free DNA replication system that we recently described (J.J. Li and T.J. Kelly, Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984; J.J. Li and T.J. Kelly, Mol. Cell. Biol. 5:1238-1246, 1985), and replication in vivo was assayed after transfection of the mutant templates into COS-1 cells. The minimal origin of replication defined by both assays included a 15-base-pair (bp) imperfect inverted repeat, a 27-bp perfect inverted repeat, and a 17-bp A/T-rich region. T-antigen binding site I was not required for DNA replication, but its presence increased replication efficiency severalfold both in vitro and in vivo. Although SP1 binding sites and enhancers had little or no effect on replication in vitro, the presence of either element markedly increased replication in vivo. Thus, the biological role of these elements is not restricted to stimulating transcription but may be more general.

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Altered intracellular transport of exogenous DNA by murine mammary tumor cell lines.

The uptake of exogenous DNA by a series of murine mammary tumor cell lines was compared with DNA uptake by normal cells and other types of transformed cells. The mammary tumor cell lines all exhibited a decreased efficiency in the transport of DNase-resistant exogenous DNA into the nuclear fraction. None of the DNA facilitators tested were able to surmount this transport defect. Normal mouse mammary gland cells, mouse embryo cells, and normal kidney cells from various species efficiently transported exogenous DNA into the nucleus. Similarly, the transport defect was not exhibited by a series of transformed cell lines, including those of mouse origin and those transformed by C-type oncornaviruses. The only exception was a murine leukemia virus-producing mouse line that displayed decreased nuclear uptake of the DNA. The nature of the exogenous DNA was apparently not a factor, since identical results were obtained with simian virus 40 and mammalian cell DNA's. The inducing agents of the mammary tumor cells also did not appear to be a factor, since cell lines derived from tumors induced spontaneously or by viral, hormonal, or chemical carcinogens all behaved similarly.

Microbial recognition and activation of plant defense systems.

Molecules released or generated during microbial entry (elicitors) are recognized by components of plant cells, ultimately resulting in the induction of a battery of plant defense responses. The molecular mechanisms underlying these signaling systems, as well as the plant defense responses they control, are becoming increasingly well characterized.

Stimulation of de novo synthesis of L-phenylalanine ammonia-lyase in relation to phytoalexin accumulation in Colletotrichum lindemuthianum elicitor-treated cell suspension cultures of french bean (Phaseolus vulgaris).

(1) The regulation of the accumulation of the isoflavonoid-derived phytoalexin phaseollin in cell suspension cultures of Dwarf French Bean (Phaseolus vulgaris/ has been investigated. (2) An elicitor preparation from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked accumulation of phaseollin in the cultures. The elicitor induced phaseollin accumulation to a level of 60% that obtained with the artificial elicitor autoclaved ribonuclease A and was maximally active at a concentration (weight basis) of at least 50 times lower than required for maximal response to ribonuclease. (3) Elicitor preparations from cell walls of Phytophthora megasperma var. sojae, a fungal pathogen of soybean, and Botrytis cinerea, the common grey mould, were much less effective than the C. lindemuthianum wall-released elicitor. (4) There was a marked but transient increase in the extractable activity of phenylalanine ammonia-lyase, the enzyme catalysing the first reaction in the biosynthesis of phaseollin from L-phenylalanine, in response to the elicitor from C. lindemuthianum. (5) Comparative density labelling with 2H from 2H2O indicated that the elicitor stimulates de novo synthesis of phenylalanine ammonie findings provide the basis of a scheme for elicitor induction of phytoalexin accumulation.

Elicitor modulation of the turnover of L-phenylalanine ammonia-lyase in French bean cell suspension cultures.

(1) The mechanisms underlying the transient increase in phenylalanine ammonia-lyase activity during phaseollin accumulation in cell suspension cultures of Dwarf French bean (Phaseolus volgaris) have been investigated using density labelling with 3H from 2H2O coupled with residual analysis of the equilibrium distribution of enzyme activity in high-resolution KBr density gradients. (2) The resolution achieved in this system is sufficient to allow quantitative analysis of the relative proportions of light, unlabelled, pre-existing enzyme and heavy, labelled, newly synthesised enzyme. (3) Elicitor released by heat treatment of cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked but transient increase in phenylalanine ammonia-lyase activity concomitant with the onset of phaseollin accumulation in the bean cultures. The induction of enzyme activity was highly dependent on elicitor concentration, with maximum induction occurring in two discrete concentration ranges; at an intermediate elicitor concentration, or at supra-optimal elicitor concentrations, no enzyme induction was observed. (4) At low concentrations of elicitor the induction of enzyme was entirely a result of elicitor stimulation of the rate of de novo enzyme production. In contrast, at higher elicitor concentrations the increase in enzyme activity was accompanied by a marked apparent stabilization of the enzyme in vivo, and the rapid but transient increase in enzyme activity was achieved by a programme of reciprocal changes in the rate constant for de novo enzyme production and the rate constant for removal of enzyme activity. Such reciprocal control of the rates of enzyme production and removal may be crucial in determining the magnitude and duration of the phytoalexin defense response. (5) Information on the specific activity of 2H label in the amino acid pools was obtained from analysis of the equilibrium distribution of residual, labelled activity. This showed directly that the turnover of the amino acid pools was fast relative to the turnover of phenylalanine ammonia-lyase and, hence, the rate of enzyme labelling was not limited by the rate of labelling of the amino acid pools. Elicitor treatment had no effect on the specific activity of label in the amino acid pools from which phenylalanine ammonia-lyase is synthesised.

Genetic approaches to the determination of structure-function relationships of G protein-coupled receptors.

The beta-adrenergic receptor (beta AR), which has been extensively characterized pharmacologically, serves as a useful model system for the analysis of the structure-function relationships of G protein-coupled receptors. Genetic and biochemical analysis has revealed that the ligand binding domain of the receptor involves residues within the hydrophobic transmembrane core of the protein. Molecular substitution experiments suggest that adrenergic agonists and antagonists are anchored to the receptor through an ionic interaction between Asp113 in the third hydrophobic region of the receptor and the protonated amine group of the ligand. In addition, catecholamine agonists are bound through hydrogen bonding interactions between two serine residues in the fifth hydrophobic domain of the receptor and the catechol hydroxyl groups of the ligand. Agonist-mediated activation of the G protein Gs requires residues within the cytoplasmic loop linking the fifth and sixth transmembrane helices which are predicted to form amphipathic alpha-helices. The strong structural similarities among G protein-coupled receptors imply that the information gained from genetic analysis of the beta AR should be applicable to other hormone and neurotransmitter receptors of this class.

Evidence for Chewing Insect-Specific Molecular Events Distinct from a General Wound Response in Leaves.

The timing of transcript accumulation of several wound-induced genes is different in insect-damaged and mechanically damaged leaves. Transcripts for the proteinase inhibitor II and 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes accumulate more rapidly in potato (Solanum tuberosum L.) leaves chewed on by caterpillars than in leaves damaged mechanically. The timing of maximum transcript accumulation was not affected by the degree of damage inflicted by the insect larvae. When applied to a mechanical wound site, regurgitant isolated from Manduca sexta larvae causes transcript accumulation profiles to shift to parallel those in insect-damaged tissue. Whether obtained from larvae fed either potato leaves or a nonplant diet, insect regurgitant fed through the petiole of detached leaves also induces accumulation of these transcripts. The transcript accumulation-inducing activity of regurgitant is enhanced by heating at 100[deg]C. Our data suggest that a heat-stable, insect-derived elicitor functions to induce the rapid accumulation of transcripts that may be involved in plant defense against herbivores. Distinct signal transduction pathways that can distinguish between insect damage and abiotic damage might therefore exist in plants.

Stress Responses in Alfalfa (Medicago sativa L.) (XIV. Changes in the Levels of Phenylpropanoid Pathway Intermediates in Relation to Regulation of L-Phenylalanine Ammonia-Lyase in Elicitor-Treated Cell-Suspension Cultures).

We have used high-resolution gas chromatography to determine the levels of trans-cinnamic acid (CA) and trans-4-coumaric acid (4CA) in alfalfa (Medicago sativa L.) cell-suspension cultures to address the role of these phenylpropanoid pathway intermediates as potential negative regulators of phenylalanine ammonia-lyase (PAL) in vivo. Exogenous addition of CA to elicitor-treated cultures resulted in rapid increases in endogenous CA, 4CA, and CA-conjugate levels associated with inhibition of the appearance of PAL transcripts. Treatment of elicited cultures with [alpha]-aminooxy-[beta]-phenylpropionic acid (AOPP), a potent and specific inhibitor of PAL activity in vivo, resulted in reductions of CA and 4CA, with concomitant increases in PAL transcripts and extractable enzyme activity. In contrast, treatment with tetcyclacis, an inhibitor of CA 4-hydroxylase, resulted in increased CA and CA-conjugate levels, decreased 4CA levels, and decreased PAL transcript levels and enzyme activity. In tetcyclasis-treated cells, the inhibition of PAL transcript appearance preceded the increase in the levels of free CA and its conjugates. In elicited cells in which the phenylpropanoid pathway was not perturbed by metabolic inhibitors, PAL transcripts accumulated rapidly and transiently, beginning to decline by 2 h postelicitation. Changes in levels of total free or conjugated CA or 4CA did not consistently correlate with these changes in transcript levels. We propose that regulation of PAL transcript levels by endogenous phenylpropanoid pathway intermediates could involve compartmentalized pools that may exist because of the microsomal localization of cinnamic acid 4-hydroxylase.

Expression and Localization of Plant Protein Disulfide Isomerase.

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.

Stress Responses in Alfalfa (XXI. Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures).

Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.). However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity. Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts. Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions. In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities. Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis.

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis.

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the enzyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from seeds of a primary transformant containing the bean PAL2 gene, which had shown homology-dependent silencing of the endogenous tobacco PAL genes. Analysis of endogenous and transgene-encoded PAL transcripts and protein in the primary transformant (T0) and first-generation (T1) overexpressor plants indicated that the transgene-encoded PAL is the cause of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T1 plants. Leaves of PAL-overexpressing plants contained increased levels of the hydroxycinnamic acid ester chlorogenic acid but not of the flavonoid rutin, indicating that PAL is the key control point for flux into chlorogenic acid. In addition, levels of the glucoside of 4-coumaric acid increased in the overexpressing plants, suggesting that the 4-coumarate:coenzyme A ligase or coumarate hydroxylase reactions might have become limiting. These results help to define the regulatory architecture of the phenylpropanoid pathway and indicate the possibility of engineering-selective changes in this complex metabolic pathway by overexpression of a single early pathway gene.

Plant natural products: the molecular genetic basis of biosynthetic diversity.

Significant advances have been made concerning the biosynthesis, regulation and genetic manipulation of plant natural products. These include insights into the structural biology of isoprenoid cyclization and polyketide condensation reactions, a better understanding of the molecular biology of plant cytochrome P450s and O-methyltransferases, and new information on the effects of natural products on human health.

Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses--a review.

In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.