RESUMO
We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven Δ12 desaturases and one of three Δ15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have Δx+3 activity, while CSFAD3A activity was exclusively at the Δ15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major Δ12 and Δ15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1(Δ9) ) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2(Δ6,9) and 18:2(Δ9,15), were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2(Δ9,15) from endogenous 18:1(Δ9) when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop.
Assuntos
Cannabis/enzimologia , Cannabis/genética , Marcação de Genes , Mutação/genética , Ácido Oleico/metabolismo , Óleos de Plantas/metabolismo , Sementes/genética , Membrana Celular/enzimologia , Temperatura Baixa , Mineração de Dados , Evolução Molecular , Ácidos Graxos Dessaturases/genética , Genes de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Microssomos/enzimologia , Sementes/metabolismo , Solubilidade , Transcriptoma/genéticaRESUMO
The STORR gene fusion event is considered essential for the evolution of the promorphinan/morphinan subclass of benzylisoquinoline alkaloids (BIAs) in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)-reticuline essential for their biosynthesis. Here, we show that of the 12 Papaver species analysed those containing the STORR gene fusion also contain promorphinans/morphinans with one important exception. P. californicum encodes a functionally conserved STORR but does not produce promorphinans/morphinans. We also show that the gene fusion event occurred only once, between 16.8-24.1 million years ago before the separation of P. californicum from other Clade 2 Papaver species. The most abundant BIA in P. californicum is (R)-glaucine, a member of the aporphine subclass of BIAs, raising the possibility that STORR, once evolved, contributes to the biosynthesis of more than just the promorphinan/morphinan subclass of BIAs in the Papaveraceae.
Assuntos
Alcaloides , Benzilisoquinolinas , Morfinanos , Papaver , Alcaloides/metabolismo , Benzilisoquinolinas/metabolismo , Fusão Gênica , Morfinanos/metabolismo , Papaver/genética , Papaver/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.
Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Expressão Gênica , Atlas como Assunto , Linhagem da Célula , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Hematopoese , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/metabolismoRESUMO
Phylogenomic analysis of whole genome sequences of five benzylisoquinoline alkaloid (BIA)-producing species from the Ranunculales and Proteales orders of flowering plants revealed the sequence and timing of evolutionary events leading to the diversification of these compounds. (S)-Reticuline is a pivotal intermediate in the synthesis of many BIAs and our analyses revealed parallel evolution between the two orders, which diverged â¼122 million years ago (MYA). Berberine is present in species across the entire Ranunculales, and we found co-evolution of genes essential for production of the protoberberine class. The benzophenanthridine class, which includes the antimicrobial compound sanguinarine, is specific to the Papaveraceae family of Ranunculales, and biosynthetic genes emerged after the split with the Ranunculaceae family â¼110 MYA but before the split of the three Papaveraceae species used in this study at â¼77 MYA. The phthalideisoquinoline noscapine and morphinan class of BIAs are exclusive to the opium poppy lineage. Ks estimation of paralogous pairs indicates that morphine biosynthesis evolved more recently than 18 MYA in the Papaver genus. In the preceding 100 million years gene duplication, neofunctionalization and recruitment of additional enzyme classes, combined with gene clustering, gene fusion, and gene amplification, resulted in emergence of medicinally valuable BIAs including morphine and noscapine.
Assuntos
Enzimas/metabolismo , Evolução Molecular , Morfina/biossíntese , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Benzofenantridinas/metabolismo , Benzilisoquinolinas/metabolismo , Alcaloides de Berberina/metabolismo , Enzimas/genética , Duplicação Gênica , Isoquinolinas/metabolismo , Morfinanos/metabolismo , Família Multigênica , Noscapina/metabolismo , Papaveraceae/genética , Filogenia , Proteínas de Plantas/genéticaRESUMO
Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for uncomplicated malaria caused by Plasmodium falciparum parasites. Other metabolites in A. annua or related species, particularly flavonoids, have been proposed to either act as antimalarials on their own or act synergistically with artemisinin to enhance antimalarial activity. We identified a mutation that disrupts the CHALCONE ISOMERASE 1 (CHI1) enzyme that is responsible for the second committed step of flavonoid biosynthesis. Detailed metabolite profiling revealed that chi1-1 lacks all major flavonoids but produces wild-type artemisinin levels, making this mutant a useful tool to test the antiplasmodial effects of flavonoids. We used whole-leaf extracts from chi1-1 and mutant lines impaired in artemisinin production in bioactivity in vitro assays against intraerythrocytic P. falciparum Dd2. We found that chi1-1 extracts did not differ from wild-type extracts in antiplasmodial efficacy nor initial rate of cytocidal action. Furthermore, extracts from the A. annua cyp71av1-1 mutant and RNAi lines impaired in amorpha-4,11-diene synthase gene expression, which are both severely compromised in artemisinin biosynthesis but unaffected in flavonoid metabolism, showed very low or no antiplasmodial activity. These results demonstrate that in vitro bioactivity against P. falciparum of flavonoids is negligible when compared to that of artemisinin.
RESUMO
Morphinan-based painkillers are derived from opium poppy (Papaver somniferum L.). We report a draft of the opium poppy genome, with 2.72 gigabases assembled into 11 chromosomes with contig N50 and scaffold N50 of 1.77 and 204 megabases, respectively. Synteny analysis suggests a whole-genome duplication at ~7.8 million years ago and ancient segmental or whole-genome duplication(s) that occurred before the Papaveraceae-Ranunculaceae divergence 110 million years ago. Syntenic blocks representative of phthalideisoquinoline and morphinan components of a benzylisoquinoline alkaloid cluster of 15 genes provide insight into how this cluster evolved. Paralog analysis identified P450 and oxidoreductase genes that combined to form the STORR gene fusion essential for morphinan biosynthesis in opium poppy. Thus, gene duplication, rearrangement, and fusion events have led to evolution of specialized metabolic products in opium poppy.
Assuntos
Benzilisoquinolinas/metabolismo , Evolução Molecular , Duplicação Gênica , Genoma de Planta , Morfinanos/metabolismo , Papaver/genética , Papaver/metabolismo , Fusão Gênica , Ordem dos Genes , Família Multigênica , NADPH-Ferri-Hemoproteína Redutase/genética , Proteínas de Plantas/genética , SinteniaRESUMO
Proteases catalyze the hydrolysis of peptide bonds in proteins/peptides inside or outside of cells. They play important roles in development and responses to environmental stresses. In arbuscular mycorrhiza (AM), symbiosis-induced protease genes were found by large-scale transcriptome analyses in different plant species, suggesting that proteolytic processes are implicated in AM. In legumes, some of these were also transcriptionally activated during the root nodule symbiosis. However, the precise function of these symbiosis-induced proteases remains unknown. Here we present a compilation of the symbiosis-induced proteases identified so far and discuss their possible roles in symbiosis.
Assuntos
Peptídeo Hidrolases/fisiologia , Raízes de Plantas/enzimologia , Simbiose/fisiologia , Indução Enzimática , Regulação da Expressão Gênica de PlantasRESUMO
Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.
Assuntos
Benzilisoquinolinas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Isoquinolinas/metabolismo , Morfinanos/metabolismo , Papaver/enzimologia , Proteínas de Plantas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Sequência de Bases , Benzilisoquinolinas/química , Sistema Enzimático do Citocromo P-450/genética , Loci Gênicos , Isoquinolinas/química , Dados de Sequência Molecular , Morfinanos/química , Mutação , Oxirredução , Papaver/genética , Proteínas de Plantas/genética , Compostos de Amônio Quaternário/químicaRESUMO
⢠The colonization of Lotus japonicus roots by the arbuscular mycorrhizal fungus Glomus intraradices was analysed in plant mutants affected in the symbiosis genes, SYM15 or SYMRK. SYMRK encodes an LRR receptor-like kinase that is, like the SYM15 gene, essential for both mycorrhizal and rhizobial symbioses. ⢠Different colonization patterns were observed in growing vs meristematically arrested roots. ⢠Three steps in the interaction were differentially impaired in the mutants: surface opening, where the anticlinal cell walls of two adjacent epidermal cells separate from each other in the vicinity of fungal hyphae; intracellular passage of hyphae through an exodermal cell and an adjacent cell of the outermost cortical layer; and arbuscule formation in cells of the two innermost cortical layers. ⢠The combined results indicate that LjSYMRK is required for the intracellular passage through exodermis and outermost cortical cell layer whereas LjSYM15 is required for surface opening and arbuscule formation.
RESUMO
Noscapine is an antitumor alkaloid from opium poppy that binds tubulin, arrests metaphase, and induces apoptosis in dividing human cells. Elucidation of the biosynthetic pathway will enable improvement in the commercial production of noscapine and related bioactive molecules. Transcriptomic analysis revealed the exclusive expression of 10 genes encoding five distinct enzyme classes in a high noscapine-producing poppy variety, HN1. Analysis of an F(2) mapping population indicated that these genes are tightly linked in HN1, and bacterial artificial chromosome sequencing confirmed that they exist as a complex gene cluster for plant alkaloids. Virus-induced gene silencing resulted in accumulation of pathway intermediates, allowing gene function to be linked to noscapine synthesis and a novel biosynthetic pathway to be proposed.
Assuntos
Antineoplásicos Fitogênicos/biossíntese , Genes de Plantas , Família Multigênica , Noscapina/metabolismo , Papaver/genética , Dados de Sequência Molecular , Papaver/enzimologia , Papaver/metabolismoRESUMO
Artemisinin is a plant natural product produced by Artemisia annua and the active ingredient in the most effective treatment for malaria. Efforts to eradicate malaria are increasing demand for an affordable, high-quality, robust supply of artemisinin. We performed deep sequencing on the transcriptome of A. annua to identify genes and markers for fast-track breeding. Extensive genetic variation enabled us to build a detailed genetic map with nine linkage groups. Replicated field trials resulted in a quantitative trait loci (QTL) map that accounts for a significant amount of the variation in key traits controlling artemisinin yield. Enrichment for positive QTLs in parents of new high-yielding hybrids confirms that the knowledge and tools to convert A. annua into a robust crop are now available.
Assuntos
Antimaláricos/metabolismo , Artemisia/genética , Artemisia/metabolismo , Artemisininas/metabolismo , Mapeamento Cromossômico , Genes de Plantas , Locos de Características Quantitativas , Cruzamentos Genéticos , DNA Complementar , Perfilação da Expressão Gênica , Estudos de Associação Genética , Humanos , Malária/tratamento farmacológico , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.
Assuntos
Autoimunidade/genética , Neoplasias da Mama/genética , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único/genética , Espondilite Anquilosante/genética , Tireoidite Autoimune/genética , Aminopeptidases/genética , Neoplasias da Mama/epidemiologia , Estudos de Casos e Controles , Mapeamento Cromossômico , Genética Populacional , Genótipo , Haplótipos/genética , Humanos , Desequilíbrio de Ligação , Antígenos de Histocompatibilidade Menor , Esclerose Múltipla/epidemiologia , América do Norte/epidemiologia , Reação em Cadeia da Polimerase , Receptores Imunológicos/genética , Receptores de Interleucina/genética , Espondilite Anquilosante/epidemiologia , Tireoidite Autoimune/epidemiologiaRESUMO
A combined genetic and transcriptome analysis was performed to study the molecular basis of the arbuscular mycorrhiza (AM) symbiosis. By testing the AM phenotype of nodulation-impaired mutants and complementation analysis, we defined seven Lotus japonicus common symbiosis genes (SYMRK, CASTOR, POLLUX, SYM3, SYM6, SYM15, and SYM24) that are required for both fungal and bacterial entry into root epidermal or cortical cells. To describe the phenotype of these mutants at the molecular level, we screened for differentiating transcriptional responses of mutant and wild-type roots by large-scale gene expression profiling using cDNA-amplified fragment length polymorphism. Two percent of root transcripts was found to increase in abundance during AM development, from which a set of AM-regulated marker genes was established. A Ser-protease (SbtS) and a Cys-protease (CysS) were also activated during root nodule development. AM-induced transcriptional activation was abolished in roots carrying mutations in common symbiosis genes, suggesting a central position of these genes in a pathway leading to the transcriptional activation of downstream genes. By contrast, AM fungus-induced gene repression appeared to be unaffected in mutant backgrounds, which indicates the presence of additional independent signaling pathways.