An Evaluation Model for the Quality of Frying Oil Using Key Aldehyde Detected by HS-GC/MS.

To establish a practical model for evaluating the oxidation of frying oil using aldehydes, the aldehydes of 10 commercial oils during frying at 180 °C were identified using headspace-gas chromatography/mass spectrometry, and the changes of common aldehydes and their correlation with carbonyl values (CV) were analyzed. The results showed that the total peak area of aldehydes increased significantly with heating time, which was related to the fatty acid and tocopherol contents of the oils. There were four common aldehydes with different trends during frying, namely, pentanal, hexanal, (E)-hept-2-enal, and nonanal. Moreover, pentanal with a high correlation with CV was selected as the quality evaluating index of frying oil due to its stable accumulation over time. Based on the linear fitting relationships between CV and pentanal, as well as the initial content ratio of linoleic acid to palmitic acid and total tocopherols in oils, a predictive model was established for evaluating the quality of frying oils with high precision and non-reagent by using mass spectrometry. In summary, this work provides theoretical support for using aldehyde as the quality evaluation index of frying oil and provides a new idea for evaluating oil deterioration from the perspective of volatile compounds.

Fluorescent labeling of the root cap cells with the bioactive NBD-S chemical probe based on the cellulose biosynthesis inhibition herbicides.

Development of the methods to examine the molecular targets of biologically active compounds is one of the most important subjects in experimental biology/biochemistry. To evaluate the usability of the (7-nitro-2,1,3-benzoxadiazole)-thioether (NBD-S) probe for this purpose, bioactive chemical probe (1) as the cellulose biosynthesis (CB) inhibitor was synthesized and tested. As a result, a variety of fluorescently-labeled particles and organelles were found in the columella root cap cells of radish plants. Of note, well-defined cellular organelles were clearly recognized in the detaching root cap cells (border-like cells). These results imply that the bioactive NBD-S chemical probe could be a valuable direct-labeling reagent. Analysis of these fluorescent substances would be helpful in providing new information on defined molecular targets and events.

Towards identification of bioactive compounds in cold vacuum extracted double cherry blossom (Gosen-Sakura) leaves.

The present research examines the possibility of finding bio-molecular compounds from the double cherry blossom (termed as 'Gosen-Sakura' of Gosen-city, Niigata-prefecture, Japan) leaves, which have been long used in the preparation of the traditional Japanese sweet (wagashi) - 'sakura-mochi'. Based on its indicated anti-microbial properties historically, our study provides a new low temperature vacuum extraction method for extracting 'near natural form of water soluble leaf (cell) extracts from the Gosen-Sakura, and demonstrates the presence of some 'novel' compound(s) with anti-tumor cell lines proliferation inhibitory affects through the MTT assay. To our knowledge, no reports exist on the sakura tree 'leaf (cell) extracts' inhibiting tumor cell line growth. We further examined and compared the effects of known compounds with anti-tumor activity, coumarin and benzyl alcohol with Gosen-Sakura leaf extract; results lead us to hypothesize that the Gosen-Sakura leaf extract contains substance(s) other than the above two known compounds, with antitumor effect. Additionally, we speculate on the underlying mechanism of action of the Gosen-Sakura leaf extract by targeting cell division at the point of DNA synthesis and causing apoptosis. In conclusion, we present scientific evidence on the presence of certain 'novel' biomolecule(s), with anti-tumor activity, in the Gosen-Sakura leaf which has been long used in Japanese sweet - the 'sakura-mochi'.

Isovaleronitrile co-induced with its precursor, l-leucine, by herbivory in the common evening primrose stimulates foraging behavior of the predatory blue shield bug.

Herbivore-induced plant volatiles play important roles in plant-insect and plant-plant interactions. The common evening primrose, Oenothera biennis, is often infested by the flea beetle, Altica oleracea, on which the predatory blue shield bug, Zicrona caerulea, is usually found. This observation suggests that the predatory bug can discriminate infested plants from intact ones to locate its prey. In this study, l-leucine-derived nitrogen-containing compounds [isovaleronitrile (3-methylbutanenitrile), (E/Z)-isovaleraldoxime and 3-methyl-1-nitrobutane] and some terpenes were identified as a characteristic volatile blend from herbivore-infested O. biennis leaves by gas chromatography/mass spectrometry, chemical synthesis, and incorporation assays using deuterium-labeled l-leucine. Volatile emission was also elicited by exogenous methyl jasmonate (MeJA), but not by mechanical damage. l-Leucine accumulated temporarily in O. biennis leaves after MeJA treatment prior to isovaleronitrile emission. Behavioral assays revealed that Z. caerulea showed a strong preference for herbivore-infested leaves, their volatiles, and isovaleronitrile in laboratory conditions.

Methyl jasmonate elicits the biotransformation of geraniol stored as its glucose conjugate into methyl geranate in Achyranthes bidentata plant.

To investigate the biotransformation pathway of airborne geraniol by Achyranthes bidentata (A. bidentata), deuterium labeled geraniol was applied with or without methyl jasmonate (MeJA), and the biosynthesized metabolites were analyzed. In A. bidentata leaves, geraniol was conjugated with glucose. The conjugate was then metabolized to afford methyl geranate only under MeJA elicitation. MeJA elicits the biotransformation of geraniol into methyl geranate by inducing the conversion of the intermediate, glucose conjugate of geraniol.

Identification of the Alarm Pheromone of Hygia lativentris and Changes in Composition during Development.

Heteropteran insects produce a series of volatile compounds from their scent glands that protect them from predators and parasites. These compounds also play roles in chemical communication that elicit aggregation, dispersal, and mating behaviors. Hygia lativentris (Coreidae) adults frequently aggregate on host plants. When disturbed, they quickly disperse with the release of a sour smell, suggesting that these bugs possess an alarm pheromone in their secretions. This adult secretion-induced dispersal has been examined with a laboratory assay. Hexanal, the predominant component of the adult secretion was identified as a component of the alarm pheromone by evaluation of the adult bug's response time and escape distance from the chemical source. Physicochemical analyses with gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy revealed that secretory components differed between nymphs and adults, and also during adult aging. Nymphs produced two unsaturated compounds, (E)-2-hexenal and (E)-4-oxo-2-hexenal, together with hexanal and 1-hexanol, which were found in all developmental stages. In adults, hexyl acetate was the major component of secretions within 3 days of emerging, while the amount of this ester decreased and those of hexanal, hexanoic acid, and hexanal trimer increased with aging. The decomposition of hexyl acetate into hexanal via 1-hexanol was attributed to the presence of esterases and alcohol dehydrogenases specifically found in adult secretory glands. In contrast, the formation of a hexanal trimer may be due to a non-enzymatic reaction under acidic conditions.

Methyl jasmonate elicits the production of methyl (E)-2-hexenoate from (Z)-2-hexenol via (Z)-2-hexenal in Achyranthes bidentata plant.

The medicinal herbal plant Achyranthes bidentata (A. bidentata) produces the sweet-odor ester - methyl (E)-2-hexenoate (1) as the major volatile in response to methyl jasmonate (MeJA). Here, we investigated the biosynthetic pathway of methyl (E)-2-hexenoate (1). The common plant precursor (Z)-3-hexenal was only slightly metabolized into methyl (E)-2-hexenoate (1), and its application scarcely enhanced the production of this ester. By contrast, a structurally related alcohol, (Z)-2-hexenol, as well as a deuteride derivative thereof could be efficiently metabolized into methyl (E)-2-hexenoate (1). Thus, we hypothesize that A. bidentata possess a specific pathway for the production of methyl (E)-2-hexenoate (1) from (Z)-2-hexenol in response to MeJA.

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection.

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Herbivore-induced phenylacetonitrile is biosynthesized from de novo-synthesized L-phenylalanine in the giant knotweed, Fallopia sachalinensis.

Plants emit a series of characteristic volatile blends when damaged by insect feeding. Phenylacetonitrile is one of the volatiles from the leaves of the giant knotweed, Fallopia sachalinensis, infested by the Japanese beetle, Popillia japonica, or treated with exogenous airborne methyl jasmonate (MeJA). We examined the precursor of the nitrile and its origin in this system. L-Phenylalanine was determined to be a precursor of the nitrile in F. sachalinensis leaves, and the phenylalanine was also induced by beetle feeding and MeJA treatment. We also found that exogenous MeJA enhanced the biosynthesis of several amino acids in F. sachalinensis leaves.

Deuterium labeling for investigating de novo synthesis of terpene volatiles in Achyranthes bidentata.

PURPOSE OF WORK: Plants synthesize and accumulate secondary metabolites as defensive volatiles against diverse stresses. We aim to unravel the jasmonate-inducible volatile de novo synthetic metabolites in plants using a deuterium-labeling technique. Jasmonic acid and its methyl ester (MeJA) are well-documented for inducing defensive volatiles. Here, we have developed an efficient deuterium oxide (D2O)-based labeling approach to determine the extent of de novo synthetic metabolites in a model plant A. bidentata bidentata. The labeling approach was demonstrated on quantitative profiling of terpene volatile organic compounds (VOCs) elicited by airborne MeJA in Achyranthes plants. We show, for the first time that airborne MeJA-elicited terpene VOCs are predominantly and differentially de novo synthesized except for a homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, which is weakly and least labelled with deuterium. D2O is therefore an efficient labeling source for investigating de novo synthetic metabolites of terpene VOCs in planta.

Quantification of jasmonic and salicylic acids in rice seedling leaves.

Jasmonic acid (JA) and salicylic acid (SA) are critical signaling components involved in various aspects of plant growth, development, and defense. Their constitutive levels vary from plant to plant and also from tissue to tissue within the same plant. Moreover, their quantitative levels change when plant is exposed to biotic and abiotic stresses. To better understand the JA- and SA-mediated signaling and metabolic pathways, it is important to precisely quantify their levels in plants/tissues/organs. However, their extraction and quantification are not trivial and still technically challenging. An effort has been made in various laboratories to develop a simple and standard procedure that can be utilized for quantification of JA and SA. Here, we present the experimental procedure and our decade of experience on extracting and quantifying them in an absolute manner in leaves of rice seedlings. We must mention that this method has been applied to both monocotyledonous and dicotyledonous plants for absolute quantification of JA and SA. As collaboration is the key towards rapid progress in science and technology, we are always open to sharing our experience in this field with any active research group with an aim to improve the procedure further and eventually to connect the importance of their (JA and SA) quantitative levels with networks of signaling and metabolic pathways in plants.

Antibacterial activity of 4-oxo-(E)-2-hexenal from adults and nymphs of the heteropteran, Dolycoris baccarum (Heteroptera: Pentatomidae).

We identified 4-oxo-(E)-2-hexenal (4-OHE) as a common component of the secretion from both Dolycoris baccarum nymphs (66.5 ± 34.7 µg/bug) and adults (87.4 ± 48.0 µg/bug) by GC/MS. We also found that this compound inhibited the growth of bacteria starting at 10 µg. The stronger antibacterial activity of 4-OHE than that of (E)-2-hexenal and (E)-2-octenal might be explained by the reactivity of α,ß-unsaturated aldehydes with nucleophilic molecules.

Methyl jasmonate is transported to distal leaves via vascular process metabolizing itself into JA-Ile and triggering VOCs emission as defensive metabolites.

Plants have developed multifaceted defensive systems against adverse environmental factors. One such recognized system is the production of metabolites in plants. Jasmonic acid (JA) and its metabolite methyl jasmonate (MeJA) are known to play key roles in metabolites production. The role of MeJA as a mobile signal has been established in Arabidopsis and Solanaceae plants. However, it remains largely unclear how MeJA-based signaling is organized via its elicited metabolites. Here, we investigated the signaling ability of MeJA by means of vascular transport using Achyranthes bidentata as an experimental plant. Results showed that MeJA was transported and essentially metabolized into its active form JA-Ile in the distal undamaged leaves accompanied by emission of volatile organic compounds. Results presented and discussed therein provide convincing evidence that MeJA acts as a transportable inter-cellular mobile compound in plants self-defense scheme.

Phenylacetonitrile from the giant knotweed, Fallopia sachalinensis, infested by the Japanese beetle, Popillia japonica, is induced by exogenous methyl jasmonate.

Phenylacetonitrile, (E)-ß-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene and (E,E)-α-farnesene were identified as Japanese beetle, Popillia japonica, feeding-induced volatiles from the leaves of the giant knotweed, Fallopia sachalinensis, but not by mechanical damage. Volatile emission was also induced by treatment with a cellular signaling molecule, methyl jasmonate. These results suggest that volatiles will be synthesized de novo by a biotic elicitor from P. japonica oral secretion.

Enzymatic synthesis of novel branched sugar alcohols mediated by the transglycosylation reaction of pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47.

Transglycosylation reactions are useful for preserving a specific sugar structure during the synthesis of branched oligosaccharides. We have previously reported a panosyl unit transglycosylation reaction by pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47 (Tonozuka et al., Carbohydr. Res., 1994, 261, 157-162). The acceptor specificity of the TVA II transglycosylation reaction was investigated using pullulan as the donor and sugar alcohols as the acceptor. TVA II transferred the α-panosyl unit to the C-1 hydroxyl group of meso-erythritol, C-1 and C-2 of xylitol, and C-1 and C-6 of d-sorbitol. TVA II differentiated between the sugar alcohols' hydroxyl groups to produce five novel non-reducing branched oligosaccharides, 1-O-α-panosylerythritol, 1-O-α-panosylxylitol, 2-O-α-panosylxylitol, 1-O-α-panosylsorbitol, and 6-O-α-panosylsorbitol. The Trp(356)→Ala mutant showed similar transglycosylation reactions; however, panose production by the mutant was 4.0-4.5-fold higher than that of the wild type. This suggests that Trp(356) is important for recognizing both water and the acceptor molecules in the transglycosylation and the hydrolysis reaction.

Conversion of airborne nerolidol to DMNT emission requires additional signals in Achyranthes bidentata.

DMNT biosynthesis was proposed to proceed via (E)-nerolidol in plants a decade ago. However, (E)-nerolidol function as airborne signal/substrate for in-vivo biosynthesis of DMNT remains to be investigated and the regulation of DMNT production and emission is largely unknown. We address both of these aspects using Achyranthes bidentata model plant in conjunction with deuterium-labeled d(5)-(E)-nerolidol, headspace, GC-FID, and GC/MS-based absolute quantification approaches. We demonstrate that airborne (E)-nerolidol is specifically metabolized in-vivo into DMNT emission, but requires airborne VOC MeJA or predator herbivore as additional environmental signal. In addition, we provide new insight into the complex regulation underlying DMNT emission, and highlight the importance of studying multiple environmental factors on emission patterns of plant VOCs and their mechanistic regulation.

Identification of a 2-cell stage specific inhibitor of the cleavage of preimplantation mouse embryos synthesized by rat hepatoma cells as 5'-deoxy-5'-methylthioadenosine.

Rat hepatoma Reuber H-35 cells produce a unique compound designated as Fr.B-25, a 2-cell stage-specific inhibitor of the cleavage of preimplantation mouse embryos cultured in vitro. Here, we identified Fr.B-25 as a purine nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), by mass spectroscopic analysis. All of the biological activities examined of authentic MTA on the development of mouse zygotes were indistinguishable from those of Fr.B-25. The mechanism of MTA action in the development of preimplantation mouse embryos was probably different from those of hypoxanthine and adenosine, which are well-characterized purine nucleosides that act as inhibitors of the cleavage of mouse 2-cell embryos. From the shared molecular and biological properties of Fr.B-25 and MTA, we concluded that Fr.B-25 is MTA. To the best of our knowledge, this is the first delineation of the effect of MTA on the development of preimplantation mammalian embryos cultured in vitro.

An in planta technique for cis-/trans-stereochemical analysis of jasmonoyl isoleucine.

A novel technique for determining the cis-/trans-stereochemistry of jasmonoyl-isoleucine by coupling its alcoholic derivatives by sodium borohydride with high performance liquid chromatography-tandem mass spectrometry is described. Resolving cis- and trans-stereochemistry of the jasmonates in Achyranthes plants exposed to airborne (exogenous) trans-d(2)MeJA was demonstrated as an example. This novel application firmly establishes for the first time that trans-d(2)MeJA is converted exclusively into trans-JA-Ile in Achyranthes leaves, whereas the subsequent de novo biosynthesized JA-Ile possesses cis-stereochemistry. The method is simple, reproducible and could be employed for in vivo cis-/trans-stereochemistry analysis of jasmonates in plants.

Rice OsACDR1 (Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance.

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

Screening for gamma-nonalactone in the headspace of freshly cooked non-scented rice using SPME/GC-O and SPME/GC-MS.

The determination of gamma-nonalactone as one of the important odor-active compounds in freshly cooked non-scented rice is reported. It was evaluated by gas chromatography-olfactometry (GC-O) analysis and identified by gas chromatography-mass spectrometry (GC-MS) analysis in the headspace above the freshly cooked non-scented rice samples extracted by using a modified headspace solid-phase microextraction (SPME) method. This component had a mass spectrum with a characteristic ion peak at m/z 85 (100%) and a linear retention index (RI) of 2,023 on a DB Wax column, consistent with those of an authentic sample of gamma-nonalactone. The odor characterization of a strong, sweet, coconut-like aroma of this compound was also validated by GC-O comparison with the authentic compound.