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Lloyd W Sumner

Publications and source records attributed to Lloyd W Sumner.

31 records · Page 2Linked to original sources
PubMed2004

A proposed framework for the description of plant metabolomics experiments and their results.

The study of the metabolite complement of biological samples, known as metabolomics, is creating large amounts of data, and support for handling these data sets is required to facilitate meaningful analyses that will answer biological questions. We present a data model for plant metabolomics known as ArMet (architecture for metabolomics). It encompasses the entire experimental time line from experiment definition and description of biological source material, through sample growth and preparation to the results of chemical analysis. Such formal data descriptions, which specify the full experimental context, enable principled comparison of data sets, allow proper interpretation of experimental results, permit the repetition of experiments and provide a basis for the design of systems for data storage and transmission. The current design and example implementations are freely available (http://www.armet.org/). We seek to advance discussion and community adoption of a standard for metabolomics, which would promote principled collection, storage and transmission of experiment data.

Database Management Systems↗
PubMed2004

Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal.

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.

Fungi↗
PubMed2004

Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus.

Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.

Amino Acid Sequence↗
PubMed2003

Metabolomics spectral formatting, alignment and conversion tools (MSFACTs).

MOTIVATION: The amplified interest in metabolic profiling has generated the need for additional tools to assist in the rapid analysis of complex data sets. RESULTS: A new program; metabolomics spectral formatting, alignment and conversion tools, (MSFACTs) is described here for the automated import, reformatting, alignment, and export of large chromatographic data sets to allow more rapid visualization and interrogation of metabolomic data. MSFACTs incorporates two tools: one for the alignment of integrated chromatographic peak lists and another for extracting information from raw chromatographic ASCII formatted data files. MSFACTs is illustrated in the processing of GC/MS metabolomic data from different tissues of the model legume plant, Medicago truncatula. The results document that various tissues such as roots, stems, and leaves from the same plant can be easily differentiated based on metabolite profiles. Further, similar types of tissues within the same plant, such as the first to eleventh internodes of stems, could also be differentiated based on metabolite profiles. AVAILABILITY: Freely available upon request for academic and non-commercial use. Commercial use is available through licensing agreement http://www.noble.org/PlantBio/MS/MSFACTs/MSFACTs.html.

Cluster Analysis↗
PubMed2003

Plant metabolomics: large-scale phytochemistry in the functional genomics era.

Metabolomics or the large-scale phytochemical analysis of plants is reviewed in relation to functional genomics and systems biology. A historical account of the introduction and evolution of metabolite profiling into today's modern comprehensive metabolomics approach is provided. Many of the technologies used in metabolomics, including optical spectroscopy, nuclear magnetic resonance, and mass spectrometry are surveyed. The critical role of bioinformatics and various methods of data visualization are summarized and the future role of metabolomics in plant science assessed.

Computational Biology↗
PubMed2003

Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis.

Soluble phenolics, wall-bound phenolics and soluble and core lignin were analyzed in transgenic alfalfa with genetically down-regulated O-methyltransferase genes involved in lignin biosynthesis. High performance liquid chromatography and principal component analysis were used to distinguish metabolic phenotypes of different transgenic alfalfa genotypes growing under standard greenhouse conditions. Principal component analysis of HPLC chromatograms did not resolve differences in leaf metabolite profiles between wild-type and transgenic plants of the same genetic background, although stem phenolic profiles were clearly different between wild-type and transgenic plants. However, the analytical methods clearly differentiated two non-transgenic alfalfa cultivars based on either leaf or stem profiles. Metabolic profiling provides a useful approach to monitoring the broader biochemical phenotypes of transgenic plants with altered expression of lignin pathway enzymes.

Cell Wall↗
PubMed2003

Regiospecific hydroxylation of isoflavones by cytochrome p450 81E enzymes from Medicago truncatula.

Mining of Medicago truncatula EST databases and screening of a root cDNA library led to the identification of three cytochrome p450 81E subfamily members. Two were functionally characterized by expression in yeast. The recombinant enzymes in yeast microsomes utilized the same isoflavone substrates, but produced different products hydroxylated at the 2' and/or 3' positions of the B-ring. When transiently expressed in alfalfa leaves, green fluorescent protein (GFP) fusions of the isoflavone 2'- and 3'-hydroxylases localized to the endoplasmic reticulum. The isoflavone 2'-hydroxylase was functional when expressed in Arabidopsis. Differential tissue-specific and biotic/abiotic stress-dependent expression patterns were observed for the isoflavone 2'-hydroxylase and 3'-hydroxylase genes, suggesting differential involvement of 2'- and 3'-hydroxylated isoflavonoids in pathogen defense and insect-induced responses, respectively, in Medicago.

Amino Acid Sequence↗
PubMed2003

Mapping the proteome of barrel medic (Medicago truncatula).

A survey of six organ-/tissue-specific proteomes of the model legume barrel medic (Medicago truncatula) was performed. Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from leaves, stems, roots, flowers, seed pods, and cell suspension cultures were obtained. Five hundred fifty-one proteins were excised and 304 proteins identified using peptide mass fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Nanoscale high-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry was used to validate marginal matrix-assisted laser desorption ionization time-of-flight mass spectrometry protein identifications. This dataset represents one of the most comprehensive plant proteome projects to date and provides a basis for future proteome comparison of genetic mutants, biotically and abiotically challenged plants, and/or environmentally challenged plants. Technical details concerning peptide mass fingerprinting, database queries, and protein identification success rates in the absence of a sequenced genome are reported and discussed. A summary of the identified proteins and their putative functions are presented. The tissue-specific expression of proteins and the levels of identified proteins are compared with their related transcript abundance as quantified through EST counting. It is estimated that approximately 50% of the proteins appear to be correlated with their corresponding mRNA levels.

Amino Acid Sequence↗
PubMed2002

Analytical and biological variances associated with proteomic studies of Medicago truncatula by two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry are being used as proteomic tools in an integrated functional genomics program focused on the model legume Medicago truncatula. Due to the perceived high levels of indeterminate error associated with 2-DE we deemed it necessary to quantify the coefficient of variance (or relative standard deviation) for both analytical and biological sources associated with 2-DE of Medicago truncatula leaf protein extracts. Leaf protein extracts were chosen because of their biological significance and due to the more challenging nature of green tissues. Analytical variance was calculated for fifty proteins from ten replicate 2-DE gels of the same protein extract. Biological variance was calculated for the same fifty proteins from ten independent 2-DE gel analyses of ten independent but similar plants grown under identical conditions. Average analytical and biological variances were calculated for both data sets and represent the average variance of approximately 500 independent measurements of protein concentration. Analytical variance was determined to be 16.2% and biological variance was determined to be 24.2%. These average variances provide a quantified and statistical basis for evaluation of protein expression changes in future comparative proteomic investigations. It is proposed that 2-DE measured protein expression levels should differ by a minimum of 3.92sigma (i.e. /+/-2sigma/ and sigma = standard deviation), or 94.7% based on our measured variances, for the difference to be significant at the 95% confidence level.

Data Interpretation, Statistical↗
PubMed2002

Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hydrogen peroxide solutions are reported for the removal of silver stain from proteins isolated in polyacrylamide gels. Removal of silver stain prior to in-gel digestion is shown to enhance sensitivity and sequence coverage of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) peptide mass maps. The rate of silver removal using H2O2 is influenced by H2O2 concentration and increases with increasing pH. The presence of complexation reagents such as ammonia from mass spectrometry compatible ammonium bicarbonate solutions enhances the efficiency and speed of H2O2-mediated silver removal. H2O2-mediated silver removal using the described procedure does not appear to have any detrimental effects on proteins but is observed to produce a slightly elevated level of methionine oxidization over that usually observed in in-gel tryptic digestion.

Bicarbonates↗
PubMed2002

Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer.

Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.

Chromatography, High Pressure Liquid↗
PubMed2001

Over-expression of cinnamate 4-hydroxylase leads to increased accumulation of acetosyringone in elicited tobacco cell-suspension cultures.

Cell-suspension cultures were produced from transgenic tobacco (Nicotiana tabacum L.) plants harboring a constitutively expressed alfalfa cinnamate 4-hydroxylase (C4H) transgene. Increased levels of C4H enzyme activity in the transgenic cultures were observed only following exposure of the cells to yeast elicitor, although alfalfa C4H transcripts were expressed at a high level from the cauliflower mosaic virus 35S promoter in the absence of elicitation. Increased expression of C4H in elicited cell-suspension cultures had no appreciable effect on the HPLC profiles of soluble phenolic compounds. However, levels of one compound, subsequently identified as 3,5-dimethoxy-4-hydroxy acetophenone (acetosyringone), were strongly elevated in the wall-bound phenolic fraction. The results are discussed in relation to the correlation between C4H activity and the synthesis of 3,5-dimethylated hydroxycinnamic acid derivatives in tobacco.

Acetophenones↗