Michael H. Beale

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at http://msi-workgroups.sourceforge.net/ or http://Msi-workgroups-feedback@lists.sourceforge.net. Further, community input related to this document can also be provided via this electronic forum.

The Strigolactone Germination Stimulants of the Plant-Parasitic Striga and Orobanche spp. Are Derived from the Carotenoid Pathway

The seeds of parasitic plants of the genera Striga and Orobanche will only germinate after induction by a chemical signal exuded from the roots of their host. Up to now, several of these germination stimulants have been isolated and identified in the root exudates of a series of host plants of both Orobanche and Striga spp. In most cases, the compounds were shown to be isoprenoid and belong to one chemical class, collectively called the strigolactones, and suggested by many authors to be sesquiterpene lactones. However, this classification was never proven; hence, the biosynthetic pathways of the germination stimulants are unknown. We have used carotenoid mutants of maize (Zea mays) and inhibitors of isoprenoid pathways on maize, cowpea (Vigna unguiculata), and sorghum (Sorghum bicolor) and assessed the effects on the root exudate-induced germination of Striga hermonthica and Orobanche crenata. Here, we show that for these three host and two parasitic plant species, the strigolactone germination stimulants are derived from the carotenoid pathway. Furthermore, we hypothesize how the germination stimulants are formed. We also discuss this finding as an explanation for some phenomena that have been observed for the host-parasitic plant interaction, such as the effect of mycorrhiza on S. hermonthica infestation.

Analysis of Detergent-Resistant Membranes in Arabidopsis. Evidence for Plasma Membrane Lipid Rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.

Secondary metabolite signalling in host-parasitic plant interactions.

The parasitic weeds Orobanche and Striga spp. are a serious threat to agriculture in large parts of the world. The lifecycle of the parasitic weeds is closely regulated by the presence of their hosts, and secondary metabolites that are produced by host plants play an important role in this interaction. Model plants, such as Arabidopsis and maize mutant collections, have been increasingly used to study these chemical signals, especially those host-produced stimulants that induce the germination of parasite seeds.

Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior

The alarm pheromone for many species of aphids, which causes dispersion in response to attack by predators or parasitoids, consists of the sesquiterpene (E)-β-farnesene (Eβf). We used high levels of expression in Arabidopsis thaliana plants of an Eβf synthase gene cloned from Mentha × piperita to cause emission of pure Eβf. These plants elicited potent effects on behavior of the aphid Myzus persicae (alarm and repellent responses) and its parasitoid Diaeretiella rapae (an arrestant response). Here, we report the transformation of a plant to produce an insect pheromone and demonstrate that the resulting emission affects behavioral responses at two trophic levels.

System-wide molecular evidence for phenotypic buffering in Arabidopsis

We profiled 162 lines of Arabidopsis for variation in transcript, protein and metabolite abundance using mRNA microarrays, two-dimensional polyacrylamide gel electrophoresis, gas chromatography time-of-flight mass spectrometry, liquid chromatography quadrupole time-of-flight mass spectrometry, and proton nuclear magnetic resonance. We added all publicly available phenotypic data from the same lines and mapped quantitative trait loci (QTL) for 40,580 molecular and 139 phenotypic traits. We found six QTL hot spots with major, system-wide effects, suggesting there are six breakpoints in a system otherwise buffered against many of the 500,000 SNPs.

Assessment of 1H NMR spectroscopy and multivariate analysis as a technique for metabolite fingerprinting of Arabidopsis thaliana

An approach to metabolite fingerprinting of crude plant extracts that utilizes 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistics has been tested. Using ecotypes of Arabidopsis thaliana as experimental material, a method has been developed for the rapid analysis of unfractionated polar plant extracts, enabling the creation of reproducible metabolite fingerprints. These fingerprints could be readily stored and compared by a variety of chemometric methods. Comparison by principal component analysis using SIMCA-P allowed the generation of residual NMR spectra of the compounds that contributed significantly to the differences between samples. From these plots, conclusions were drawn with respect to the identity and relative levels of metabolites differing between samples.

Plant Metabolomics: The Missing Link in Functional Genomics Strategies

After the establishment of technologies for high-throughput DNA sequencing (genomics), gene expression analysis (transcriptomics), and protein analysis (proteomics), the remaining functional genomics challenge is that of metabolomics . Metabolomics is the term coined for essentially comprehensive,

Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts

A functional assay for gibberellin (GA) receptors is described based on the induction of α-amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA4 immobilised to Sepharose beads. A 17-thiol derivative of GA4, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA4-17-Sepharose induces high levels of α-amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA4. In order to eliminate the possibility that GA4 may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA4, GA4-17-Sepharose and control Sepharose estimated by determining the relative amounts of α-amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA417-Sepharose induces α-amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.

Transcriptional Adaptation of Mycosphaerella graminicola to Programmed Cell Death (PCD) of Its Susceptible Wheat Host

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.