Brome mosaic virus Infection of Rice Results in Decreased Accumulation of RNA1.

Brome mosaic virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 replication.

Metabolome analysis of Ephedra plants with different contents of ephedrine alkaloids by using UPLC-Q-TOF-MS.

Metabolome analysis of four varieties of Ephedra plants, which contain different amounts of ephedrine alkaloids, was demonstrated in this study. The metabolites were comprehensively analyzed by using ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and the ephedrine alkaloids were also profiled. Subsequently, multivariate analyses of principal component analysis (PCA) and batch-learning self-organizing mapping (BL-SOM) analysis were applied to the raw data of the total ion chromatogram (TIC). PCA was performed to visualize the fingerprints characteristic for each Ephedra variant and the independent metabolome clusters were formed. The metabolite fingerprints were also visualized by BL-SOM analysis and were displayed as a lattice of colored individual cells which was characteristic for each Ephedra variant. BL-SOM analysis was also used for identification of chemical marker peaks because the information assigned to a cell represented either increases or decreases in peak intensities. Using this analysis, ephedrine alkaloids were successfully selected from the TICs as chemical markers for each Ephedra variant and this result suggested that BL-SOM analysis was an effective method for the selection of marker metabolites. We report our study here as a practical case of metabolomic study on medicinal resources.

Differential gene expression profiles of red and green forms of Perilla frutescens leading to comprehensive identification of anthocyanin biosynthetic genes.

Differential screening by PCR-select subtraction was carried out for cDNAs from leaves of red and green perilla, two chemovarietal forms of Perilla frutescens regarding anthocyanin accumulation. One hundred and twenty cDNA fragments were selected as the clones preferentially expressed in anthocyanin-accumulating red perilla over the nonaccumulating green perilla. About half of them were the cDNAs encoding the proteins related presumably to phenylpropanoid-derived metabolism. The cDNAs encoding glutathione S-transferase (GST), PfGST1, and chalcone isomerase (CHI), PfCHI1, were further characterized. The expression of PfGST1 in an Arabidopsis thaliana tt19 mutant lacking the GST-like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in tt19, indicating a function of PfGST1 in vacuole sequestration of anthocyanin in perilla. The recombinant PfCHI1 could stereospecifically convert naringenin chalcone to (2S)-naringenin. PfGST1 and PfCHI1 were preferentially expressed in the leaves of red perilla, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. These results suggest that the genes of the whole anthocyanin biosynthetic pathway are regulated in a coordinated manner in perilla.

Metabolite annotations based on the integration of mass spectral information.

A large number of metabolites are found in each plant, most of which have not yet been identified. Development of a methodology is required to deal systematically with unknown metabolites, and to elucidate their biological roles in an integrated 'omics' framework. Here we report the development of a 'metabolite annotation' procedure. The metabolite annotation is a process by which structures and functions are inferred for metabolites. Tomato (Solanum lycopersicum cv. Micro-Tom) was used as a model for this study using LC-FTICR-MS. Collected mass spectral features, together with predicted molecular formulae and putative structures, were provided as metabolite annotations for 869 metabolites. Comparison with public databases suggests that 494 metabolites are novel. A grading system was introduced to describe the evidence supporting the annotations. Based on the comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that are frequently found in tomato fruit tissues, and predicted novel metabolic pathways for flavonoids and glycoalkaloids. These results demonstrate that metabolite annotation facilitates the systematic analysis of unknown metabolites and biological interpretation of their relationships, which provide a basis for integrating metabolite information into the system-level study of plant biology.

Differential metabolomics unraveling light/dark regulation of metabolic activities in Arabidopsis cell culture.

Differential metabolomics based on a non-targeted FT-ICR/MS analysis demonstrated metabolite accumulation patterns reflecting light/dark conditions in Arabidopsis T87 cell culture. First, FT-ICR/MS data sets were converted into metabolome information using the Dr.DMASS software (http://kanaya.naist.jp/DrDMASS/). A quick search of a metabolite-species database, KNApSAcK (http://kanaya.naist.jp/KNApSAcK/), was implemented to assign metabolite candidates to each accurate MS data (<1 ppm) through the prediction of molecular formulas, and the candidate structures were further studied using MS/MS analyses. Specific metabolites representing the culture conditions included sugars, phenylpropanoid derivatives, flavonol aglycons, and a plastid nonmevalonate pathway intermediate. Transcriptomics data were obtained in parallel and analyzed using a transcriptome analysis tool, KaPPA-View (http://kpv.kazusa.or.jp/kappa-view/). The specific accumulation patterns of flavonol aglycons were in good agreement with the light/dark regulation of a cytochrome P450 gene, CYP75B, and the build-up of 2-C-methyl-D-erythritol 4-phosphate, a nonmevalonate pathway intermediate, in the light grown cells was also consistent with a gene expression profile. The differential metablomics scheme based on the FT-ICR/MS metabolomics can serve as an evaluation system of metabolic activities contributing to successful identification and proper manipulation of key enzymatic steps in metabolic engineering studies.

Loss of anthocyanins in red-wine grape under high temperature.

To determine the mechanism of inhibition of anthocyanin accumulation in the skin of grape berries due to high temperature, the effects of high temperature on anthocyanin composition and the responses in terms of gene transcript levels were examined using Vitis vinifera L. cv. Cabernet Sauvignon. High temperature (maximum 35 degrees C) reduced the total anthocyanin content to less than half of that in the control berries (maximum 25 degrees C). HPLC analysis showed that the concentrations of anthocyanins, with the exception of malvidin derivatives (3-glucoside, 3-acetylglucoside, and 3-p-coumaroylglucoside), decreased considerably in the berries grown under high temperature as compared with the control. However, Affymetrix Vitis GeneChip microarray analysis indicated that the anthocyanin biosynthetic genes were not strongly down-regulated at high temperature. A quantitative real time PCR analysis confirmed this finding. To demonstrate the possibility that high temperature increases anthocyanin degradation in grape skin, stable isotope-labelled tracer experiments were carried out. Softened green berries of Cabernet Sauvignon were cut and aseptically incubated on filter paper with 1 mM aqueous L-[1-(13)C]phenylalanine solution for 1 week. Thereafter, the changes in (13)C-labelled anthocyanins were examined under different temperatures (15, 25, and 35 degrees C). In the berries cultured at 35 degrees C, the content of total (13)C-labelled anthocyanins that were produced before exposure to high temperature was markedly reduced as compared with those cultured at 15 degrees C and 25 degrees C. These data suggest that the decrease in anthocyanin accumulation under high temperature results from factors such as anthocyanin degradation as well as the inhibition of mRNA transcription of the anthocyanin biosynthetic genes.

Convergent evolution in the BAHD family of acyl transferases: identification and characterization of anthocyanin acyl transferases from Arabidopsis thaliana.

Members of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines.

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and beta-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean.

Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.

Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.

Elucidation of gene-to-gene and metabolite-to-gene networks in arabidopsis by integration of metabolomics and transcriptomics.

Since the completion of genome sequences of model organisms, functional identification of unknown genes has become a principal challenge in biology. Post-genomics sciences such as transcriptomics, proteomics, and metabolomics are expected to discover gene functions. This report outlines the elucidation of gene-to-gene and metabolite-to-gene networks via integration of metabolomics with transcriptomics and presents a strategy for the identification of novel gene functions. Metabolomics and transcriptomics data of Arabidopsis grown under sulfur deficiency were combined and analyzed by batch-learning self-organizing mapping. A group of metabolites/genes regulated by the same mechanism clustered together. The metabolism of glucosinolates was shown to be coordinately regulated. Three uncharacterized putative sulfotransferase genes clustering together with known glucosinolate biosynthesis genes were candidates for involvement in biosynthesis. In vitro enzymatic assays of the recombinant gene products confirmed their functions as desulfoglucosinolate sulfotransferases. Several genes involved in sulfur assimilation clustered with O-acetylserine, which is considered a positive regulator of these genes. The genes involved in anthocyanin biosynthesis clustered with the gene encoding a transcriptional factor that up-regulates specifically anthocyanin biosynthesis genes. These results suggested that regulatory metabolites and transcriptional factor genes can be identified by this approach, based on the assumption that they cluster with the downstream genes they regulate. This strategy is applicable not only to plant but also to other organisms for functional elucidation of unknown genes.

Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.

The integration of metabolomics and transcriptomics can provide precise information on gene-to-metabolite networks for identifying the function of unknown genes unless there has been a post-transcriptional modification. Here, we report a comprehensive analysis of the metabolome and transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene encoding an MYB transcription factor, for the identification of novel gene functions involved in flavonoid biosynthesis. For metabolome analysis, we performed flavonoid-targeted analysis by high-performance liquid chromatography-mass spectrometry and non-targeted analysis by Fourier-transform ion-cyclotron mass spectrometry with an ultrahigh-resolution capacity. This combined analysis revealed the specific accumulation of cyanidin and quercetin derivatives, and identified eight novel anthocyanins from an array of putative 1800 metabolites in PAP1 over-expressing plants. The transcriptome analysis of 22,810 genes on a DNA microarray revealed the induction of 38 genes by ectopic PAP1 over-expression. In addition to well-known genes involved in anthocyanin production, several genes with unidentified functions or annotated with putative functions, encoding putative glycosyltransferase, acyltransferase, glutathione S-transferase, sugar transporters and transcription factors, were induced by PAP1. Two putative glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1 expression were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic activity of their recombinant proteins in vitro and results of the analysis of anthocyanins in the respective T-DNA-inserted mutants. The functional genomics approach through the integration of metabolomics and transcriptomics presented here provides an innovative means of identifying novel gene functions involved in plant metabolism.