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1.
Proc Natl Acad Sci U S A ; 117(2): 1174-1180, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882449

RESUMO

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.


Assuntos
Alcaloides/biossíntese , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Indolizidinas/metabolismo , Policetídeo Sintases/metabolismo , Fosfato de Piridoxal/metabolismo , Alcaloides/genética , Alcaloides/isolamento & purificação , Antibacterianos/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Catálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Hidroxilação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Família Multigênica , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos , Fosfato de Piridoxal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
J Appl Genet ; 60(3-4): 269-281, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31410824

RESUMO

White lupin (Lupinus albus L.) is a legume grain crop cultivated since ancient Greece and Egypt. Modern white lupin cultivars are appreciated as a source of protein with positive nutraceutical impact. However, white lupins produce anti-nutritional compounds, quinolizidine alkaloids, which provide bitter taste and have a negative influence on human health. During domestication of this species, several recessive alleles at unlinked loci controlling low alkaloid content were selected. One of these loci, pauper, was exploited worldwide providing numerous low-alkaloid cultivars. However, molecular tracking of pauper has been hampered due to the lack of diagnostic markers. In the present study, the synteny-based approach was harnessed to target pauper locus. Single-nucleotide polymorphisms flanking pauper locus on white lupin linkage map as well as candidate gene sequences elucidated from the narrow-leafed lupin (L. angustifolius L.) chromosome segment syntenic to the pauper linkage group region were transformed to PCR-based molecular markers. These markers were analyzed both in the mapping population and world germplasm collection. From fourteen markers screened, eleven were localized at a distance below 1.5 cM from this locus, including five co-segregating with pauper. The linkage of these markers was confirmed by high LOD values (up to 58.4). Validation performed in the set of 127 bitter and 23 sweet accessions evidenced high applicability of one marker, LAGI01_35805_F1_R1, for pauper locus selection, highlighted by the low ratio of false-positive scores (2.5%). LAGI01_35805 represents a homolog of L. angustifolius acyltransferase-like (LaAT) gene which might hypothetically participate in the alkaloid biosynthesis process in lupins.


Assuntos
Alcaloides/genética , Marcadores Genéticos/genética , Lupinus/genética , Mapeamento Cromossômico , Ligação Genética , Genoma de Planta/genética , Folhas de Planta/genética , Sintenia/genética
3.
Genes (Basel) ; 10(6)2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167507

RESUMO

Low-alkaloid content is an important breeding target to improve the quality of lupin seeds. An APETALA2/ethylene response transcription factor, RAP2-7, is likely a candidate gene for the major alkaloid locus iucundus, and plays a crucial role in regulation of seed alkaloid content in narrow-leafed lupin (NLL; Lupinus angustifolius L.). Here, we exploited a single-nucleotide polymorphism within RAP2-7 credibly associated with seed alkaloid content, to develop the co-dominant derived cleaved amplified polymorphic sequence (dCAPS) marker iuc_RAP2-7. Marker validation in 202 NLL accessions demonstrated that seed alkaloid content ≥0.9% of the seed dry weight was associated with the high-alkaloid marker band (Iucundus genotypes), whereas alkaloid content up to 0.5% of the seed dry weight was associated with the low-alkaloid marker band (iucundus genotypes). Within a given detection limit, iuc_RAP2-7 unambiguously identified all but three low-alkaloid accessions. The latter accessions apparently have a different regulatory mechanism for seed alkaloid content because the RAP2-7 gene/putative promoter sequence and expression of alkaloid-associated genes in the leaves of the three ambiguous accessions were similar to those of bitter Iucundus lines. We consider the iuc_RAP2-7 marker is a powerful tool that will facilitate NLL marker-assisted selection by rapid rejection of bitter Iucundus genotypes and thus accelerate development of new low-alkaloid cultivars.


Assuntos
Alcaloides/genética , Lupinus/genética , Folhas de Planta/genética , Sementes/genética , Alcaloides/metabolismo , Proteínas de Arabidopsis , Biomarcadores/metabolismo , Amplificação de Genes , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta , Genótipo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lupinus/metabolismo , Desenvolvimento Vegetal/genética , Folhas de Planta/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Sementes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Microb Pathog ; 132: 335-342, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31100407

RESUMO

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.


Assuntos
Bacillus/fisiologia , Candida albicans/patogenicidade , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade , Lepidópteros/imunologia , Alcaloides/genética , Alcaloides/metabolismo , Alcaloides/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/fisiologia , Contagem de Colônia Microbiana , Defensinas/genética , Defensinas/metabolismo , Defensinas/farmacologia , Modelos Animais de Doenças , Expressão Gênica/genética , Hemócitos/imunologia , Hemócitos/metabolismo , Hemolinfa , Interações entre Hospedeiro e Microrganismos/genética , Sistema Imunitário , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Larva/imunologia , Larva/microbiologia , Lepidópteros/genética , Lepidópteros/microbiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacologia , Quinolinas/metabolismo , Quinolinas/farmacologia , Esporos Bacterianos , Taxa de Sobrevida
5.
Mol Biotechnol ; 61(7): 498-505, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31119478

RESUMO

Taxoid 10ß-O-acetyltransferase (DBAT) is the key enzyme to produce baccatin III, a key precursor in paclitaxel synthesis, by acetyl group transfer from acetyl-CoA to the C10 hydroxyl of 10-deacetylbaccatin III. In this study, the recombinant DBAT (rDBAT) was immobilized by cross-linked enzyme aggregates (CLEAs). To further optimize the enzyme recovery, single-factor experiment and response surface methodology were applied. 60% ammonium sulfate as precipitant, 0.05% glutaraldehyde as fixing agent, pH 7.0, 2 h as cross-linking time, 30 °C as cross-linking temperature were confirmed to be the optimum conditions to prepare the CLEAs-rDBAT in single-factor experiment. In addition, 62% for ammonium sulfate saturation, 0.15% for glutaraldehyde, and pH 6.75 were confirmed to be the optimum conditions with averagely 73.9% activity recovery in 3 replications, which was consistent with the prediction of response surface methodology. After cross-linking, the optimum temperature of CLEAs-rDBAT rose up to 70 °C and CLEAs-rDBAT could be recycled for three times.


Assuntos
Acetiltransferases/biossíntese , Alcaloides/biossíntese , Enzimas Imobilizadas/biossíntese , Proteínas Recombinantes/biossíntese , Acetiltransferases/genética , Alcaloides/genética , Reagentes para Ligações Cruzadas , Paclitaxel/biossíntese , Taxoides
7.
BMC Genomics ; 20(1): 7, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30611193

RESUMO

BACKGROUND: Celastrus angulatus Maxim is a kind of crucial and traditional insecticidal plant widely distributed in the mountains of southwest China. Celangulin V is the efficient insecticidal sesquiterpenoid of C. angulatus and widely used in pest control in China, but the low yield and discontinuous supply impeded its further popularization and application. Fortunately, the development of synthetic biology provided an opportunity for sustainable supply of Celangulin V, for which understanding its biosynthetic pathway is indispensable. RESULTS: In this study, six cDNA libraries were prepared from leaf and root of C. angulatus before global transcriptome analyses using the BGISEQ-500 platform. A total of 104,950 unigenes were finally obtained with an average length of 1200 bp in six transcriptome databases of C. angulatus, in which 51,817 unigenes classified into 25 KOG classifications, 39,866 unigenes categorized into 55 GO functional groups, and 48,810 unigenes assigned to 135 KEGG pathways, 145 of which were putative biosynthetic genes of sesquiterpenoid and triterpenoid. 16 unigenes were speculated to be related to Celangulin V biosynthesis. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis. CONCLUSIONS: This study is the first report on transcriptome analysis of C. angulatus, and 16 unigenes probably involved in the biosynthesis of Celangulin V were finally collected. The transcriptome data will make great contributions to research for this specific insecticidal plant and the further gene mining for biosynthesis of Celangulin V and other sesquiterpene polyol esters.


Assuntos
Celastrus/genética , Haptenos/genética , Anotação de Sequência Molecular , Transcriptoma/genética , Alcaloides/genética , Vias Biossintéticas/genética , Celastrus/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Haptenos/biossíntese , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento
8.
Molecules ; 24(1)2019 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-30621230

RESUMO

The genus Uncaria is an important source of traditional Chinese medicines with multiple therapeutic effects. The identification of the correct species and accurate determination of the contents of bioactive constituents are important for quality control of Uncaria medicinal materials. Here, an integrated evaluation system based on DNA barcoding for species identification and quantitative analysis by LC-MS/MS has been established. DNA barcoding based on the ITS2 barcode region showed sufficient discriminatory power to precisely identify 24 samples from seven Uncaria species. The length of the 24 ITS2 sequences of Uncaria samples is 227 bp, and 17 variation sites were detected. Additionally, the results of qualitative and quantitative chemical analyses by LC-MS/MS indicated that the chemical compositions of all Uncaria samples were similar; while their contents of targeted alkaloids in samples from different species and origin areas were different. The contents of rhynchophylline (RC) and isorhynchophylline (IRC) were 2.9⁻1612 mg/kg and 2.60⁻1299 mg/kg in all tested samples, respectively. This study concludes that DNA barcoding should be used as the first screening step for Uncaria medicinal materials. Then, integration of DNA barcoding with chemical analyses should be applied in quality control of Uncaria medicinal materials in the medicinal industry.


Assuntos
Alcaloides/genética , Código de Barras de DNA Taxonômico , DNA de Plantas/genética , Uncaria/genética , Alcaloides/química , Cromatografia Líquida , DNA de Plantas/química , Alcaloides Indólicos/química , Medicina Tradicional Chinesa , Espectrometria de Massas em Tandem , Uncaria/química
9.
Mol Genet Genomics ; 294(2): 315-328, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30443676

RESUMO

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.


Assuntos
Endófitos/genética , Epichloe/genética , Compostos Heterocíclicos com 2 Anéis/metabolismo , Poaceae/genética , Poliaminas/metabolismo , Alcaloides/genética , Animais , Resistência à Doença/genética , Epichloe/crescimento & desenvolvimento , Edição de Genes , Controle Biológico de Vetores , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Poaceae/microbiologia , Reprodução Assexuada/genética , Simbiose/genética , Gorgulhos/genética , Gorgulhos/patogenicidade
10.
Nicotine Tob Res ; 21(7): 991-995, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29401309

RESUMO

The Food and Drug Administration has announced the potential for mandated lowering of nicotine levels in combustible cigarettes. The World Health Organization has recommended a lowering of cigarette filler nicotine levels to below 0.4 mg/g. To devise appropriate nicotine control strategies, regulators must consider technical feasibility, timelines for compliance, and potential impediments to implementation. Outlined here is previously unsummarized information on genetic approaches that might be used to reduce nicotine levels in cured tobacco leaves. For the benefit of regulators, altered alkaloid or toxicant profiles that might result by implementation of some of these methodologies are discussed. Also mentioned are potential licensing or regulatory impediments to use of some of the technologies per se. Implications: An understanding of technical feasibility of plant-based nicotine reduction technologies, along with the potential for corresponding alterations in alkaloid or toxicant profiles, is needed by regulators to develop effective nicotine control strategies with minimal impediments or undesirable consequences.


Assuntos
Alcaloides/efeitos adversos , Nicotina/efeitos adversos , Produtos do Tabaco/efeitos adversos , United States Food and Drug Administration , Organização Mundial da Saúde , Alcaloides/genética , Engenharia Genética/métodos , Engenharia Genética/tendências , Humanos , Nicotina/genética , Estados Unidos , United States Food and Drug Administration/tendências
11.
Genomics ; 111(6): 1360-1372, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30248489

RESUMO

Crown imperial (CI) has been used in traditional medicine. Today it is known that such beneficial effects are due to its richness in steroidal alkaloids (SA). Using de novo transcriptomics, orthologues/paralogues finder, phylogenetic analysis and tissue- and developmental stage-specific expression analysis, we identified ten genes and several TFs involved in the biosynthesis of SA in CI. The comparative analysis of ten genes expression profiles revealed the possibility of their co-regulation, which may imply the possibility of their organization in metabolic gene clusters. Having in mind convergent evolution of steroidal biosynthetic pathways in flowering plants and records of convergent evolution of specific proteins, observed expression patterns open a reasonable interest to investigate the possibility of the existence of genes cluster organization in SA pathway in the family Liliaceae or at least in some species of genus Fritillaria. Obtained results support transcriptomics as useful approach in elucidating genes underlying complex biochemical pathways.


Assuntos
Alcaloides/biossíntese , Fritillaria/genética , Fritillaria/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Esteroides/biossíntese , Transcriptoma , Alcaloides/genética , Vias Biossintéticas , Biologia Computacional , Fritillaria/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/metabolismo , Esteroides/metabolismo
12.
Biomolecules ; 8(4)2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30326637

RESUMO

To increase the understanding of alkaloid biosynthesis in Nicotiana tabacum during whole plant growth periods, variations of the contents of alkaloids and the transcription of key biosynthetic genes in fresh leaves were investigated in three varieties at five developmental stages. Six alkaloids were analyzed by gas chromatograph⁻mass spectrometry (GC⁻MS) and the most abundant alkaloid was observed during the upper leaves maturing stage in the varieties, among which the alkaloid content of K326 was the highest. Considering the genetic effect, variance analysis indicated that the developmental stage played a predominant role in alkaloid accumulation. Moreover, the levels of biosynthetic gene transcripts in the leaves at the vigorous growing stage might contribute to the contents of alkaloids in the leaves during the maturing stages. To further illuminate the metabolism of alkaloid biosynthesis, a correlation among alkaloids was also documented.


Assuntos
Alcaloides/genética , Tabaco/genética , Transcrição Genética , Alcaloides/química , Alcaloides/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Tabaco/química , Tabaco/crescimento & desenvolvimento
13.
Curr Microbiol ; 75(12): 1649-1654, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30267141

RESUMO

Two strains of Pseudomonas putida, Psp-LUP and Psp-SPAR, capable of growth on the quinolizidine alkaloids, lupanine and sparteine respectively, were studied here. We report the isolation of Psp-SPAR and the complete genome sequencing of both bacteria. Both were confirmed to belong to P. putida, Psp-LUP close to the type isolate of the species (NBRC14164T) and Psp-SPAR close to strains KT2440 and F1. Psp-SPAR did not grow on lupanine but did contain a gene encoding a putative quinolizidine-17-hydroxylase peptide which exhibited high similarity (76%identity) to the lupanine-17-hydroxylase characterised from Psp-LUP.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Pseudomonas putida/genética , Esparteína/genética , Esteroide 17-alfa-Hidroxilase/genética , Alcaloides/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Análise de Sequência/métodos , Esparteína/análogos & derivados , Sequenciamento Completo do Genoma/métodos
14.
Biol Pharm Bull ; 41(12): 1870-1873, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30259882

RESUMO

In traditional Tibetan medicinal system, Berberis herbs mainly originate from the dried barks of Berberis kansuensis, Berberis dictyophylla, Berberis diaphana, and Berberis vernae. In this study, molecular phylogenetic method based on four markers (i.e., rbcL, internal transcribed spacer (ITS), ITS2, and psbA-trnH) and HPLC chemical analysis were used to evaluate the chemical and genetic differences between the four Berberis species. The results showed that the discriminatory power of ITS, ITS2 and psbA-trnH was low, but the rbcL marker was highly effective and reliable for the species differentiation. The four Berberis species can be successfully classified based on phylogenetic analysis of the rbcL sequences. Moreover, the results of chemical analysis showed that four main alkaloids (i.e., berberine, palmatine, magnoflorine, and jatrorrhizine) cannot be used as chemical markers for discrimination of the four Berberis species. These findings provide valuable information for distinguishing the four Berberis Tibetan herbs.


Assuntos
Alcaloides/análise , Alcaloides/genética , Berberis/química , Berberis/genética , Filogenia , Preparações de Plantas/química , Cromatografia Líquida de Alta Pressão/métodos , Plantas Medicinais/química , Plantas Medicinais/genética
15.
J Biosci ; 43(2): 391-405, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29872026

RESUMO

Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperative morphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT, COR, T6ODM, and CODM genes in different tissues of Papaver somniferum (i.e. root, bottom part of stem, upper part of stem, leaf, capsule wall, and capsule content) over five distinguished ontogenic stages (i.e. rosette, bud initiation, pendulous bud, flowering, and lancing). Apart from bottom stem and leaf, the maximum thebaine content occurred in lancing stage, while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times. Regarding codeine, excepting upper stem, the highest ratios of codeine were observed at flowering and lacing stages, while negligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart from upper stem, it appears that late ontogeneic times including lancing and flowering are the most appropriate phases to achieve high amounts of morphine, while at early stages the aforesaid alkaloid possessed lower accumulation. Furthermore, all the five genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various sampling times. Interestingly, a connection occurred between transcript ratio of SalAT and thebaine content, suggesting that thebaine biosynthesis is coordinated tightly by the enzymatic function of SalAT enzyme. Meanwhile, despite low magnitudes of T6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine and morphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from the producing (source) tissues to the roots (sink), known as a phenomenon of 'source-to-sink transportation'. The results, altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporal oscillations on the magnitudes of all the above-mentioned alkaloids alongside transcription ratios of the key genes in opium poppy.


Assuntos
Alcaloides/genética , Dopa Descarboxilase/genética , Morfina/química , Papaver/genética , Proteínas de Plantas/genética , Tirosina Descarboxilase/genética , Alcaloides/química , Flores/química , Flores/genética , Regulação da Expressão Gênica de Plantas , Morfina/metabolismo , Papaver/química , Folhas de Planta/química , Folhas de Planta/genética , Caules de Planta/química , Caules de Planta/genética
16.
J Exp Bot ; 68(20): 5527-5537, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29155974

RESUMO

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis.


Assuntos
Alcaloides/genética , Carboxiliases/genética , Regulação da Expressão Gênica de Plantas , Lupinus/genética , Proteínas de Plantas/genética , Transcriptoma , Alcaloides/biossíntese , Carboxiliases/metabolismo , Lupinus/metabolismo , Proteínas de Plantas/metabolismo , Quinolizidinas/metabolismo
17.
Phytochemistry ; 141: 1-10, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28535420

RESUMO

Plants have developed defensive specialized metabolites over the course of evolution. In the genus Hordeum, which includes the important cereal crop barley, specialized metabolites such as hordatines, benzoxazinones, and gramine have been identified. Hordeum species are classified into four clades, H, Xu, Xa, and I. The presence or absence of defensive specialized metabolites was analyzed in representative Hordeum species that included all of the four clades. In the H clade, Hordeum vulgare accumulated hordatines but not benzoxazinones, whereas H. bulbosum accumulated neither compound. Some accessions in the H clade accumulated gramine. Species in the clades I and Xa accumulated benzoxazinones without hordatines. In H. murinum, a Xu clade species, neither hordatines nor benzoxazinones were detected. Two hitherto undescribed compounds were found to commonly accumulate in H. bulbosum in the H clade and H. murinum in the Xu clade. On the basis of spectroscopic analyses, they were identified as dehydrodimers of feruloylagmatine and were designated murinamides A and B. Radical coupling reactions with feruloylagmatine as a substrate by peroxidase afforded murinamides A and B. These compounds showed antifungal activities against Bipolaris sorokiniana and Fusarium asiaticum, indicating their defensive roles. Because hordatines are also dehydrodimers of hydroxycinnamic acid amides (HCAAs) of agmatine, both the H and Xu clade species are considered to accumulate the same class of compounds. Thus, when the H/Xu clades split from the I/Xa clades during evolution, the defensive metabolites shifted from benzoxazinones to dehydrodimers of agmatine HCAAs plus gramine in the H/Xu clades.


Assuntos
Evolução Molecular , Hordeum/metabolismo , Compostos Fitoquímicos/metabolismo , Alcaloides/genética , Alcaloides/metabolismo , Benzoxazinas/metabolismo , Ácidos Cumáricos/metabolismo , Hordeum/genética , Alcaloides Indólicos , Compostos Fitoquímicos/genética
18.
Chem Commun (Camb) ; 53(19): 2826-2829, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28225144

RESUMO

Heterologous expressions and purifications of all WelU proteins from the welwitindolinone pathways in Hapalosiphon welwitschii UTEX B1830 and IC-52-3 led to the discovery that WelU1 and WelU3 selectively assemble 12-epi-fischerindole U (2) and 12-epi-hapalindole C (1), respectively, from 3-geranyl 3-isocyanovinyl indolenine (4) via an enzymatic cascade featuring the Cope rearrangement, stereoselective aza-Prins cyclization and regioselective carbocation deposition. In combination with the in vitro characterization of WelU1/WelU3-homolog AmbU4 for the biogenesis of 12-epi-hapalindole U, this study provide a unified view on the origin of the early stage structural diversifications in hapalindole-type alkaloid biosynthesis, post common intermediate 4.


Assuntos
Alcaloides/biossíntese , Alcaloides/genética , Alcaloides/química , Estrutura Molecular , Estereoisomerismo
19.
Genome ; 60(6): 496-509, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28177829

RESUMO

Symbiotic associations between tall fescue grasses and asexual Epichloë fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass (Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs.


Assuntos
Alcaloides/genética , Endófitos/genética , Epichloe/genética , Poaceae/genética , Sequência de Bases , Variação Genética/genética , Genômica/métodos , Genótipo
20.
Genet Mol Res ; 15(3)2016 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-27525873

RESUMO

An F3 population consisting of 117 F2:3 families derived from a cross between two varieties of rice, Gongmi No. 3 and Diantun 502, with a large difference in their resistant starch and total alkaloid content, was used for quantitative trait locus (QTL) mapping. Two QTLs of resistant starch for rice (qRS7-1, qRS7-2) were identified in a linkage group on chromosome 7, which could explain phenotypic variance from 7.6 to 17.3%, due to additive effects for resistant starch from Gongmi No. 3 or over-dominance effects for qRS7-2 of the marker interval (RM3404-RM478) on chromosome 7 from Gongmi No. 3, accounting for 13.8-17.3% of the phenotypic variance. Two QTLs of total alkaloids for brown rice (qALb7-1, qALb7-2) were identified in the same linkage group, which could explain phenotypic variance from 7.7 and 19.3%, respectively, due to dominance or over-dominance effects for total alkaloids on chromosome 7 from Diantun 502. To our knowledge, these are the first QTLs to be identified, which are related to resistant starch and total alkaloid content in rice. These results are beneficial for understanding the genetic basis of, as well as for developing markers linked with, resistant starch and total alkaloids of functional components for marker-assisted selection breeding in rice.


Assuntos
Cromossomos de Plantas/genética , Oryza/genética , Locos de Características Quantitativas , Alcaloides/genética , Alcaloides/metabolismo , Mapeamento Cromossômico , Cruzamentos Genéticos , Genes de Plantas , Estudos de Associação Genética , Ligação Genética , Loci Gênicos , Marcadores Genéticos , Oryza/metabolismo , Fenótipo , Melhoramento Vegetal , Amido/genética , Amido/metabolismo
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