Brian D. McGarvey

Isoflavonoid biosynthesis and accumulation in developing soybean seeds

Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pods, and in elicitor-treated or pathogen-challenged tissues. Incorporation of radiolabel into isoflavonoids was observed when developing embryos and other plant organs were fed with [14C]phenylalanine. Embryos excised from developing soybean seeds also accumulated isoflavonoids from a synthetic medium. A maternal effect on seed isoflavonoid content was noted in reciprocal crosses between soybean cultivars that differ in seed isoflavonoids. From these results, we propose that developing soybean embryos have an ability to synthesize isoflavonoids de novo, but that transport from maternal tissues may in part contribute to the accumulation of these natural products in the seed.

High-performance liquid chromatographic methods for the determination of N-methylcarbamate pesticides in water, soil, plants and air

Abstract High-performance liquid chromatographic methods of analysis for 31 N-methylcarbamate pesticides and 46 of their metabolises in water, soil, plant and air samples are reviewed. Consideration is given to extraction, clean-up, chromatographic separation and detection techniques including UV absorbance, fluorescence, electrochemical and mass spectrometric detection.

High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.

Reduced translocation of cadmium from roots is associated with increased production of phytochelatins and their precursors

Cadmium (Cd) is a non-essential trace element and its environmental concentrations are approaching toxic levels, especially in some agricultural soils. Understanding how and where Cd is stored in plants is important for ensuring food safety. In this study, we examined two plant species that differ in the distribution of Cd among roots and leaves. Lettuce and barley were grown in nutrient solution under two conditions: chronic (4 weeks) exposure to a low, environmentally relevant concentration (1.0 μM) of Cd and acute (1 h) exposure to a high concentration (5.0 mM) of Cd. Seedlings grown in solution containing 1.0 μM CdCl₂ did not show symptoms of toxicity and, at this concentration, 77% of the total Cd was translocated to leaves of lettuce, whereas only 24% of the total Cd was translocated to barley leaves. We tested the hypothesis that differential accumulation of Cd in roots and leaves is related to differential concentrations of phytochelatins (PCs), and its precursor peptides. The amounts of PCs and their precursor peptides in the roots and shoots were measured using HPLC. Each of PC₂₋₄ was synthesized in the barley root upon chronic exposure to Cd and did not increase further upon acute exposure. In the case of lettuce, no PCs were detected in the root given either Cd treatment. The high amounts of PCs produced in barley root could have contributed to preferential retention of Cd in barley roots.

Comparative evaluation of eight software programs for alignment of gas chromatography–mass spectrometry chromatograms in metabolomics experiments

Since retention times of compounds in GC-MS chromatograms always vary slightly from chromatogram to chromatogram, it is necessary to align chromatograms before comparing them in metabolomics experiments. Several software programs have been developed to automate this process. Here we report a comparative evaluation of the performance of eight programs using prepared samples of mixtures of chemicals, and an extract of tomato vines spiked with three concentrations of a mixture of alkanes. The programs included in the comparison were SpectConnect, MetaboliteDetector 2.01a, MetAlign 041012, MZmine 2.0, TagFinder 04, XCMS Online 1.21.01, MeltDB and GAVIN. Samples were analyzed by GC-MS, chromatograms were aligned using the selected programs, and the resulting data matrices were preprocessed and submitted to principal components analysis. In the first trial, SpectConnect, MetAlign and MetaboliteDetector correctly identified ≥90% of the true positives. In the second trial, MetAlign and MetaboliteDetector correctly identified 87% and 81% of the true positives, respectively. In addition, in both trials >90% of the peaks identified by MetAlign and MetaboliteDetector were true positives.

DIMINUTO 1 affects the lignin profile and secondary cell wall formation in Arabidopsis

Brassinosteroids (BRs) play a crucial role in plant growth and development and DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis, was previously identified as a cell elongation factor in Arabidopsis thaliana. Through promoter expression analysis, we showed that DIM1 was expressed in most of the tissue types in seedlings and sectioning of the inflorescence stem revealed that DIM1 predominantly localizes to the xylem vessels and in the interfascicular cambium. To investigate the role of DIM1 in cell wall formation, we generated loss-of-function and gain-of-function mutants. Disruption of the gene function caused a dwarf phenotype with up to 38 and 23% reductions in total lignin and cellulose, respectively. Metabolite analysis revealed a significant reduction in the levels of fructose, glucose and sucrose in the loss-of-function mutant compared to the wild type control. The loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type, but no changes were detected in the gain-of-function mutant. Phloroglucinol and toluidine blue staining showed a size reduction of the vascular apparatus with smaller and disintegrated xylem vessels in the inflorescence stem of the loss-of-function mutant. Taken together, these data indicate a role for DIM1 in secondary cell wall formation. Moreover, this study demonstrated the potential role of BR hormones in modulating cell wall structure and composition.

EVIDENCE FOR AN ISOBUTYLAMIDE ASSOCIATED WITH HOST-PLANT RESISTANCE TO WESTERN FLOWER THRIPS, Frankliniella occidentalis, IN CHRYSANTHEMUM

Bioassay-directed fractionation of extracts of chrysanthemum leaves using a choice test permitted isolation of a fraction that exhibited repellent activity against the western flower thrips (WFT). Analysis of this fraction from cultivars exhibiting varying degrees of host-plant resistance to WFT by high performance liquid chromatography revealed a distinctive peak, the height of which correlated with the degree of resistance of those cultivars to WFT. The peak was attributed to a novel unsaturated isobutylamide, N-isobutyl-(E, E, E, Z)-2,4,10,12-tetradecatetraen-8-ynamide.

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry.

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization-multiple stage tandem mass spectrometry (ESI-MS(n)) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na](+) and [M + NH(4)](+) ions, a characteristic [M-glycosyl + H](+) ion was observed in full-scan mode with in-source fragmentation. Sequential in-trap collision-induced dissociation of [M + Na](+) ions from 11,12-diesters revealed consistent preferred losses of substituents first from C-12, then from C-11, followed by losses of monosaccharide fragments from the C-3 tri- and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high-performance liquid chromatography coupled to ESI-MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C(4)H(7)O C-11/C-12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in-source fragmentation and in-trap collision-induced dissociation and supports the usefulness of LC-ESI-MS(n) not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds.

The Impact of Greenhouse Tomato (Solanales: Solanaceae) Floral Volatiles on Bumble Bee (Hymenoptera: Apidae) Pollination

ABSTRACT Greenhouse tomato (Lycopersicon esculentum Mill) production relies on pollination by commercially-produced bumble bee (Bombus impatiens Cresson) colonies. Inadequate pollination by bumble bees has been a problem for growers at certain times of year; however, its cause has yet to be determined. Bumble bees have been shown to exit tomato greenhouses to forage on flowers of other plants. This study investigates tomatos floral characteristics and their affect on bumble bee pollination by 1) observing foraging preferences for bumble bees on greenhouse tomato, 2) determining if the plants floral advertisements could be used by the bees to estimate pollen availability, and 3) identifying temporal changes in floral display which correspond to peak bumble bee activity. Flower size (petal length, anther cone width, and anther cone length) and floral scent (release of &bgr;-phellandrene, 2-carene, &agr;-pinene, and p-cymene) were evaluated to identify the pollinator-important characteristics of tomato flowers. Our results indicate that 1) bumble bees preferred to pollinate flowers which produce less &bgr;-phellandrene and 2-carene in comparison to flowers producing more of these volatiles, 2) flower size and floral scent are not likely used by the bees to estimate pollen availability, and 3) cultivars are inconsistent in their production of floral volatiles during peak bumble bee activity, &bgr;-phellandrene and 2-carene may be antiherbivory volatiles and reduced production during peak bee activity may help to facilitate pollination of tomato. Pollinator-repellent volatiles may help to protect flowers from damage caused by over-pollination.

Comparative metabolomic analysis of Saccharomyces cerevisiae during the degradation of patulin using gas chromatography–mass spectrometry

A comparative metabolomic analysis was conducted on Saccharomyces cerevisiae cells with and without patulin treatment using gas chromatography–mass spectrometry-based approach. A total of 72 metabolites were detected and compared, including 16 amino acids, 29 organic acids and alcohols, 19 sugars and sugar alcohols, 2 nucleotides, and 6 miscellaneous compounds. Principle component analysis showed a clear separation of metabolome between the cells with and without patulin treatment, and most of the identified metabolites contributed to the separation. A close examination of the identified metabolites showed an increased level of most of the free amino acids, an increased level of the intermediates in the tricarboxylic acid cycle, a higher amount of glycerol, a changed fatty acid composition, and a decreased level of cysteine and glutathione in the cells with patulin treatment. This finding indicated a slower protein synthesis rate and induced oxidative stress in the cells with patulin treatment, and provided new insights into the effect of toxic chemicals on the metabolism of organisms.