An Optimized SPME-GC-MS Method for Volatile Metabolite Profiling of Different Alfalfa (Medicago sativa L.) Tissues.

Solid-phase microextraction (SPME) was coupled to gas chromatography mass spectrometry (GC-MS) and a method optimized to quantitatively and qualitatively measure a large array of volatile metabolites in alfalfa glandular trichomes isolated from stems, trichome-free stems, and leaves as part of a non-targeted metabolomics approach. Major SPME extraction parameters optimized included SPME fiber composition, extraction temperature, and extraction time. The optimized SPME method provided the most chemically diverse coverage of alfalfa volatile and semi-volatile metabolites using a DVB/CAR/PDMS fiber, extraction temperature of 60 °C, and an extraction time of 20 min. Alfalfa SPME-GC-MS profiles were processed using automated peak deconvolution and identification (AMDIS) and quantitative data extraction software (MET-IDEA). A total of 87 trichome, 59 stem, and 99 leaf volatile metabolites were detected after background subtraction which removed contaminants present in ambient air and associated with the fibers and NaOH/EDTA buffer solution containing CaCl2. Thirty-seven volatile metabolites were detected in all samples, while 15 volatile metabolites were uniquely detected only in glandular trichomes, 9 only in stems, and 33 specifically in leaves as tissue specific volatile metabolites. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of glandular trichomes, stems, and leaves showed that the volatile metabolic profiles obtained from the optimized SPME-GC-MS method clearly differentiated the three tissues (glandular trichomes, stems, and leaves), and the biochemical basis for this differentiation is discussed. Although optimized using plant tissues, the method can be applied to other types of samples including fruits and other foods.

Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins.

INTRODUCTION: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. OBJECTIVES: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. METHODS: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. RESULTS: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of ß-amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2ß-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2ß-hydroxyoleanolic acid, hederagenin and total saponin levels. CONCLUSIONS: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2ß-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.

Integrated metabolomics and transcriptomics reveal enhanced specialized metabolism in Medicago truncatula root border cells.

Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased ß-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4'-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4'-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously.

Suppression of plant defense responses by extracellular metabolites from Pseudomonas syringae pv. tabaci in Nicotiana benthamiana.

BACKGROUND: Pseudomonas syringae pv. tabaci (Pstab) is the causal agent of wildfire disease in tobacco plants. Several pathovars of Pseudomonas syringae produce a phytotoxic extracellular metabolite called coronatine (COR). COR has been shown to suppress plant defense responses. Interestingly, Pstab does not produce COR but still actively suppresses early plant defense responses. It is not clear if Pstab produces any extracellular metabolites that actively suppress early defense during bacterial pathogenesis. RESULTS: We found that the Pstab extracellular metabolite extracts (Pstab extracts) remarkably suppressed stomatal closure and nonhost hypersensitive response (HR) cell death induced by a nonhost pathogen, P. syringae pv. tomato T1 (Pst T1), in Nicotiana benthamiana. We also found that the accumulation of nonhost pathogens, Pst T1 and P. syringae pv. glycinea (Psgly), was increased in N. benthamiana plants upon treatment with Pstab extracts . The HR cell death induced by Pathogen-Associated Molecular Pattern (INF1), gene-for-gene interaction (Pto/AvrPto and Cf-9/AvrCf-9) and ethanol was not delayed or suppressed by Pstab extracts. We performed metabolite profiling to investigate the extracellular metabolites from Pstab using UPLC-qTOF-MS and identified 49 extracellular metabolites from the Pstab supernatant culture. The results from gene expression profiling of PR-1, PR-2, PR-5, PDF1.2, ABA1, COI1, and HSR203J suggest that Pstab extracellular metabolites may interfere with SA-mediated defense pathways. CONCLUSIONS: In this study, we found that Pstab extracts suppress plant defense responses such as stomatal closure and nonhost HR cell death induced by the nonhost bacterial pathogen Pst T1 in N. benthamiana.

Long-lifetime water-washable ceramic catalyst filter for air purification.

Particulate matter (PM) and volatile organic compounds (VOCs) are recognised as hazardous air pollutants threatening human health. Disposable filters are generally used for air purification despite frequent replacement and waste generation problems. However, the development of a novel regenerable and robust filter for long-term use is a huge challenge. Here, we report on a new class of facile water-washing regenerable ceramic catalyst filters (CCFs), developed to simultaneously remove PM (>95%) and VOCs (>82%) in single-pass and maximized space efficiency by coating the inner and outer filter channels with an inorganic membrane and a Cu2O/TiO2 photocatalyst, respectively. The CCFs reveal four-fold increase in the maximum dust loading capacity (approximately 20 g/L) in relation to conventional filters (5 g/L), and can be reused after ten regeneration capability with simple water washing retaining initial PM and VOC removal performances. Thus, the CCFs can be well-suited for indoor and outdoor air purification for 20 years, which shows a huge increase in lifetime compared to the 6-month lifespan of conventional filters. Finally, we believe that the development and implementation of CCFs for air purification can open new avenues for sustainable technology through renewability and zero-waste generation.

TrichOME: a comparative omics database for plant trichomes.

Plant secretory trichomes have a unique capacity for chemical synthesis and secretion and have been described as biofactories for the production of natural products. However, until recently, most trichome-specific metabolic pathways and genes involved in various trichome developmental stages have remained unknown. Furthermore, only a very limited amount of plant trichome genomics information is available in scattered databases. We present an integrated "omics" database, TrichOME, to facilitate the study of plant trichomes. The database hosts a large volume of functional omics data, including expressed sequence tag/unigene sequences, microarray hybridizations from both trichome and control tissues, mass spectrometry-based trichome metabolite profiles, and trichome-related genes curated from published literature. The expressed sequence tag/unigene sequences have been annotated based upon sequence similarity with popular databases (e.g. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Transporter Classification Database). The unigenes, metabolites, curated genes, and probe sets have been mapped against each other to enable comparative analysis. The database also integrates bioinformatics tools with a focus on the mining of trichome-specific genes in unigenes and microarray-based gene expression profiles. TrichOME is a valuable and unique resource for plant trichome research, since the genes and metabolites expressed in trichomes are often underrepresented in regular non-tissue-targeted cDNA libraries. TrichOME is freely available at http://www.planttrichome.org/.

Characterization and discrimination of premium-quality, waxy, and black-pigmented rice based on odor-active compounds.

BACKGROUND: Odor-active compounds have been studied in cooked aromatic rice, but not in specialty rice types that have distinctly different flavors. We analyzed the odor-active compounds emanating from three different types of specialty rice (premium-quality, waxy and black-pigmented) and identified the differences in odor-active compounds among them. RESULTS: Twenty-one, 21 and 23 odorants were detected using GC-O for cooked samples of premium-quality, waxy and black-pigmented rice cultivars, respectively. Hexanal was the main odorant in premium-quality and waxy cultivars; however, waxy cultivars had 16 times higher hexanal odor activity values (OAVs) than premium-quality cultivars, indicating premium-quality rice had a less pronounced overall aroma. 2-Acetyl-1-pyrroline was the main contributor to overall aroma in black-pigmented rice, followed by guaiacol. The three types of specialty rice were clearly discriminated based on the OAVs of their odor-active compounds using multivariate analyses. Six odor-active compounds [2-acetyl-1-pyrroline, guaiacol, hexanal, (E)-2-nonenal, octanal and heptanal] contributed the most in discriminating the three types of specialty rice. Six very similar superior cultivars of premium rice could likewise be readily separated using aroma chemistry. CONCLUSION: The ability to discriminate the aroma among rice types using the OAVs of the principal odor-active compounds facilitates our understanding of the aroma chemistry of specialty rice.

Large-Scale Profiling of Saponins in Different Ecotypes of Medicago truncatula.

A total of 1,622 samples representing 201 Medicago truncatula ecotypes were analyzed using ultrahigh pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) to ascertain saponin profiles in different M. truncatula ecotypes and to provide data for a genome-wide association study and subsequent line selection for saponin biosynthesis. These ecotypes originated from 14 different Mediterranean countries, i.e., Algeria, Cyprus, France, Greece, Israel, Italy, Jordan, Libya, Morocco, Portugal, Spain, Syria, Tunisia, and Turkey. The results revealed significant differences in the saponin content among the ecotypes. European ecotypes generally contained higher saponin content than African ecotypes (p < 0.0001). This suggests that M. truncatula ecotypes modulate their secondary metabolism to adapt to their environments. Significant differences in saponin accumulation were also observed between the aerial and the root tissues of the same ecotypes (p < 0.0001). While some saponins were found to be present in both the aerial and root tissues, zanhic acid glycosides were found predominantly in the aerial tissues. Bayogenin and hederagenin glycosides were found mostly in roots. The differential spatially resolved accumulation of saponins suggests that saponins in the aerial and root tissues play different roles in plant fitness. Aerial saponins such as zanhic glycosides may act as animal feeding deterrent and root saponins may protect against soil microbes.

Comparison of odor-active compounds from six distinctly different rice flavor types.

Using a dynamic headspace system with Tenax trap, GC-MS, GC-olfactometry (GC-O), and multivariate analysis, the aroma chemistry of six distinctly different rice flavor types (basmati, jasmine, two Korean japonica cultivars, black rice, and a nonaromatic rice) was analyzed. A total of 36 odorants from cooked samples were characterized by trained assessors. Twenty-five odorants had an intermediate or greater intensity (odor intensity >or= 3) and were considered to be major odor-active compounds. Their odor thresholds in air were determined using GC-O. 2-Acetyl-1-pyrroline (2-AP) had the lowest odor threshold (0.02 ng/L) followed by 11 aldehydes (ranging from 0.09 to 3.1 ng/L), guaiacol (1.5 ng/L), and 1-octen-3-ol (2.7 ng/L). On the basis of odor thresholds and odor activity values (OAVs), the importance of each major odor-active compound was assessed. OAVs for 2-AP, hexanal, ( E)-2-nonenal, octanal, heptanal, and nonanal comprised >97% of the relative proportion of OAVs from each rice flavor type, even though the relative proportion varied among samples. Thirteen odor-active compounds [2-AP, hexanal, ( E)-2-nonenal, octanal, heptanal, nonanal, 1-octen-3-ol, ( E)-2-octenal, ( E, E)-2,4-nonadienal, 2-heptanone, ( E, E)-2,4-decadienal, decanal, and guaiacol] among the six flavor types were the primary compounds explaining the differences in aroma. Multivariate analysis demonstrated that the individual rice flavor types could be separated and characterized using these compounds, which may be of potential use in rice-breeding programs focusing on flavor.

Characterization of volatile aroma compounds in cooked black rice.

Black rice ( Oryza sativa L.), an aromatic specialty rice popular in Asia, has a unique flavor, the volatile chemistry of which has not been reported. The objectives of this research were to study volatile profiles of cooked black rice and to characterize the odor-active compounds. Thirty-five volatile compounds were identified by gas chromatography-mass spectrometry using a dynamic headspace system with Tenax trapping. Aldehydes and aromatics were quantitatively in the greatest abundance, accounting for 80.1% of total relative concentration of volatiles. The concentration of 2-acetyl-1-pyrroline (2-AP) was high, exceeded only by hexanal, nonanal, and 2-pentylfuran. A total of 25 odor-active compounds, determined by gas chromatography-olfactometry, were applied to principal component analysis, demonstrating significant differences between a black and a traditional white rice cultivar in terms of aroma and explaining 93.0% of the total variation. 2-AP, guaiacol, indole, and p-xylene largely influenced the difference between the aroma in cooked black and white rice. 2-AP and guaiacol were major contributors to the unique character of black rice based on odor thresholds, relative concentrations, and olfactometry.