Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

<p>Abstract</p> <p>Background</p> <p><it>Papaver somniferum </it>(opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.</p> <p>Results</p> <p>A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database.</p> <p>Conclusions</p> <p>The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.</p

Fatty acid composition of developing sea buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature seed

Background Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18∶2ω-6) and α-linolenic (18∶3ω-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16∶1ω-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology. Results GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33–36% and 30–36%, respectively, while the pulp oil contained palmitoleic acid at 32–42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein>nucleic acid>carbohydrate>lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry. Conclusion This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1∶1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions

Gene expression analysis of flax seed development

<p>Abstract</p> <p>Background</p> <p>Flax, <it>Linum usitatissimum </it>L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.</p> <p>Results</p> <p>We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions <ext-link ext-link-id="LIBEST_026995" ext-link-type="gen">LIBEST_026995</ext-link> to <ext-link ext-link-id="LIBEST_027011" ext-link-type="gen">LIBEST_027011</ext-link>) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for <it>in silico </it>expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.</p> <p>Conclusions</p> <p>We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.</p

Fatty Acid Composition of Developing Sea Buckthorn (Hippophae rhamnoides L.) Berry and the Transcriptome of the Mature Seed

Background: Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18:2\u3c9-6) and \u3b1-linolenic (18:3\u3c9-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16:1\u3c9-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology. Results: GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and \u3b1-linolenic acids at 33-36% and 30-36%, respectively, while the pulp oil contained palmitoleic acid at 32-42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein>nucleic acid>carbohydrate>lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry. Conclusion: This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and \u3b1-linolenic acid in a close to 1:1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions.Peer reviewed: YesNRC publication: Ye

Maximizing the Efficacy of SAGE Analysis Identifies Novel Transcripts in Arabidopsis

The efficacy of using Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of the model dicotyledonous plant Arabidopsis was assessed. We describe an iterative tag-to-gene matching process that exploits the availability of the whole genome sequence of Arabidopsis. The expression patterns of 98% of the annotated Arabidopsis genes could theoretically be evaluated through SAGE and using an iterative matching process 79% could be identified by a tag found at a unique site in the genome. A total of 145,170 reliable experimental tags from two Arabidopsis leaf tissue SAGE libraries were analyzed, of which 29,632 were distinct. The majority (93%) of the 12,988 experimental tags observed greater than once could be matched within the Arabidopsis genome. However, only 78% were matched to a single locus within the genome, reflecting the complexities associated with working in a highly duplicated genome. In addition to a comprehensive assessment of gene expression in Arabidopsis leaf tissue, we describe evidence of transcription from pseudo-genes as well as evidence of alternative mRNA processing and anti-sense transcription. This collection of experimental SAGE tags could be exploited to assist in the on-going annotation of the Arabidopsis genome

Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy

Recent advances in DNA sequencing technology and analytical mass spectrometry are providing unprecedented opportunities to develop the functional genomics resources required to investigate complex biological processes in non-model plants. Opium poppy produces a wide variety of benzylisoquinoline alkaloids (BIAs), including the pharmaceutical compounds codeine, morphine, noscapine and papaverine. A functional genomics platform to identify novel BIA biosynthetic and regulatory genes in opium poppy has been established based on the differential metabolite profile of eight selected cultivars. Stem cDNA libraries from each of the eight opium poppy cultivars were subjected to 454 pyrosequencing and searchable expressed sequence tag databases were created from the assembled reads. These deep and integrated metabolite and transcript databases provide a nearly complete representation of the genetic and metabolic variances responsible for the differential occurrence of specific BIAs in each cultivar as demonstrated using the biochemically well characterized pathway from tyrosine to morphine. Similar correlations between the occurrence of specific transcripts and alkaloids effectively reveals candidate genes encoding uncharacterized biosynthetic enzymes as shown using cytochromes P450 potentially involved in the formation of papaverine and noscapine.Peer reviewed: YesNRC publication: Ye

Wheat transcriptome profiling reveals abscisic and gibberellic acid treatments regulate early-stage phytohormone defense signaling, cell wall fortification, and metabolic switches following <i>Fusarium graminearum</i>-challenge

ABSTRACTBackgroundApplication of the wheat phytohormones abscisic acid (ABA) or gibberellic acid (GA) affect Fusarium head blight (FHB) disease severity; however, the molecular underpinnings of the elicited phenotypes remain unclear. Herein, the transcriptomic responses of an FHB-susceptible wheat cultivar ‘Fielder’ were characterized upon treatment with ABA, an ABA receptor antagonist (AS6), or GA in the presence or absence of Fusarium graminearum (Fg) challenge.ResultsA total of 30,876 differentially expressed genes (DEGs) where identified in ‘Fielder’ (26,004) and Fg (4,872). Fg challenge alone resulted in the most substantial wheat DEGs contributing to 57.2% of the total transcriptomic variation. Using a combination of topology overlap and correlation analyses, 9,689 Fg-related wheat DEGs were defined. Further enrichment analysis of the top 1% networked wheat DEGs identified critical expression changes within defense responses, cell structural metabolism, molecular transport, and membrane/lipid metabolism. Fg-challenged conditions also included the expression of a putative Fg ABA-biosynthetic cytochrome P450 and repression of wheat FUS3 for dysregulating ABA and GA crosstalk. ABA treatment alone elicited 4536 (32%) wheat DEGs common to those of the Fg-challenge, and Fg+ABA further enhanced 888 (12.5%) of them. These ABA elicited DEGs are involved in defense through both classical and non-classical phytohormone signaling and regulating cell wall structures including polyphenolic metabolism. Conversely, Fg+GA opposed 2239 (33%) Fg-elicited wheat DEGs, including modulating primary and secondary metabolism, defense responses, and flowering genes. ABA and jointly ABA⍰Fg⍰[Fg+ABA] treatments repressed, while Fg+GA induced an over-representation of wheat DEGs mapping to chromosome 6BL. Finally, compared to Fg+ABA, co-application of Fg+AS6 did not antagonize ABA biosynthesis or signal but rather elicited antagonistic Fg (557) and wheat (11) DEGs responses directly tied to stress responses, phytohormone transport, and FHB.ConclusionsComparative transcriptomics highlight the effects of wheat phytohormones on individual pathway and global metabolism simultaneously. Application of ABA may reduce FHB severity through misregulating defense mechanisms and cell wall fortification pathways. GA application may alter primary and secondary metabolism, creating a metabolic shift to ultimately reduce FHB severity. By comparing these findings to those previously reported for four additional plant genotypes, an additive model of the wheat-Fg interaction is proposed.</jats:sec